WO2016058094A1

WO2016058094A1 - BIOSENSOR BASED ON GβΥ-INTERACTING PROTEINS TO MONITOR G-PROTEIN ACTIVATION

Info

Publication number: WO2016058094A1
Application number: PCT/CA2015/051032
Authority: WO
Inventors: Michel Bouvier; Christian Le Gouill; Mireille HOGUE; Viktoriya LUKASHEVA
Original assignee: Université de Montréal
Priority date: 2014-10-14
Filing date: 2015-10-14
Publication date: 2016-04-21
Also published as: EP3207052B1; AU2015333543A1; IL250949A0; JP2017536104A; CN107074926A; PL3207052T3; IL250949B; NZ729735A; ES2832331T3; CA2963579C; CA2963579A1; JP6932636B2; US10877036B2; EP3207052A4; EP3207052A1; AU2015333543B2; DK3207052T3; SG11201701895VA; US20170234870A1; CN107074926B

Abstract

Resonance energy transfer (RET)- or protein-fragment complement assay (PCA)- based biosensors useful for assessing the activity of G-proteins are described. These biosensors are based on the competition between the Gα subunit and a Gβγ interacting protein (βγ IP) for the binding to the Gβγ dimer. These biosensors comprises (1) a βγ IP and (2) a Gβ or Gγ protein; a GPCR; or a plasma membrane targeting domain, fused to suitable RET or PCA tags. Methods using such biosensors for different applications, including the identification of agents that modulates G-protein activity or for the characterization of GPCR signaling/regulation, such as G-protein preferences and activation profiles of GPCRs, are also described.

Description

BIOSENSOR BASED ON G y-INTERACTING PROTEINS TO MONITOR G-PROTEIN

ACTIVATION

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application serial No. 62/063,622, filed on October 14, 2014, which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present disclosure relates to the monitoring of G-protein activation, and more specifically to a signalling biosensor for detecting G-protein activation.

BACKGROUND ART

Hetero-trimeric G-proteins consisting of three subunits α, β and γ, relays the information provided by G-protein-coupled receptors (GPCRs) to various intracellular effectors. In the absence of stimulation, the a-subunit of the G-protein is in complex with a GDP (guanosine diphosphate) molecule. The conformational change that follows receptor activation by a ligand, promotes the phosphorylation of the GDP molecule into a GTP (guanosine triphosphate). The GTP-bound Ga subunit dissociates from the ΰβγ subunits, both of which are then available to interact with downstream effectors and modulate their activity. G-protein activation can thus be assessed by analyzing those downstream effectors through their interaction with ΰβγ, using Gpy interacting proteins (βγΙΡ). Following GTP hydrolysis to GDP by the Ga subunit, the Ga affinity for Gpy is restored and the three subunits re-associate to form an inactive hetero-trimeric G-protein, ending the engagement of effectors and thus signal transduction (Gilman 1987).

In addition to the classical activation of G-proteins by GPCRs, other proteins can also modulate the activity of these hetero-trimeric G-proteins, such as regulators of G-protein signalling (RGS), activators of G-protein signalling (AGS), and resistance to inhibitors of cholinesterase 8 proteins (Ric-8). In some of these non-canonical signalling pathways, the guanine exchange factor (GEF) activity classically exerted by GPCRs is replaced by another protein such as Ric-8 for example (Boularan and Kehrl, 2014).

G-protein-coupled receptor kinases (GRKs) 2 and 3, which were first characterized for their role in desensitization of receptors, are also effectors engaged through their interaction with G y subunits. GRK2 and GRK3 contain a pleckstrin homology (PH) domain that interacts with the Gpy subunits of G-proteins, upon their dissociation from the activated GTP-bound Ga subunit (Pitcher, Inglese et al. 1992) (Touhara, Inglese et al. 1994). As a consequence, proteins interacting with ΰβγ (βγΙΡ) such as GRK2 and GRK3, can be used to directly study G-protein activation by GPCRs or other G-protein activators. Several approaches are currently used in the drug discovery industry to assess the activation of GPCRs and thus the engagement of G-proteins by receptors, such as calcium mobilization assay or radioactive assay based on GTPyS incorporation by G-proteins. The calcium mobilization assay measures a signaling event occurring downstream Gq activation and can be applied to Gi or Gs-coupled receptors only when coupled with the use of modified Ga subunits. In the case of GTPyS incorporation assay, the activation of the various hetero-trimeric G-proteins is directly measured on cell membranes using radioactive GTP_Y ³⁵S, and cannot be performed in living cells.

The activation of G-proteins in living cells, without modifying the G-protein activator or the Ga subunit, has thus not been explored so far. Furthermore, the known methods are not suitable to study all the different G-proteins using the same detection partners. Such assays would be particularly useful in the different stages of the drug discovery process, by enabling characterization of G-protein coupling profile and facilitating the identification of new compounds with defined signalling properties for use in screening assays and structure-activity relationship studies, for example. This is particularly true given the importance of G-protein activators as drug targets, with 26% of all prescribed medications acting through GPCRs (Garland 2013) . Even though several approaches are available to support the development of new therapeutically active molecules targeting G-protein activators, the discovery of novel drugs is often limited by the dearth of information available on the precise mechanism of action of those compounds.

There is thus a need for novel tools and assays to assess activation of G-proteins. The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

The present invention provides the following items 1 to 68:

1 . A biosensor system for detecting G-protein activity, said biosensor system comprising the elements defined in (A) or (B):

(A)

(i) a first biosensor comprising:

a first component comprising a Gpy interacting protein (βγΙΡ) fused to (a) a resonance energy transfer (RET) donor; (b) a RET acceptor or (c) a first fragment of a reporter protein; and

a second component comprising a fused Gp protein or a fused Gy protein, wherein said Gp protein or said Gy protein is fused to (a) a RET donor; (b) a RET acceptor or (c) a second fragment of said reporter protein;

(ii) a second biosensor comprising: the first and second components defined in (i); and

a third component comprising a recombinant Ga protein;

wherein (a) if said βγΙΡ is fused to said RET donor, said Gp or Gy protein is fused to said RET acceptor; (b) if said βγΙΡ is fused to said RET acceptor, said Gp or Gy protein is fused to said RET donor; and (c) if said βγΙΡ is fused to said first fragment of said reporter protein, said Gp or Gy protein is fused to said second fragment of said reporter protein; or

(B)

(i) a biosensor comprising

a first component comprising a Gpy interacting protein (βγΙΡ) fused to (a) a RET donor; (b) a RET acceptor or (c) a first fragment of a reporter protein;

a second component comprising a fused G-protein coupled receptor (GPCR), wherein said GPCR is fused at its C-terminal to (a) a RET donor; (b) a RET acceptor or (c) a second fragment of said reporter protein;

a third component comprising a recombinant Ga protein;

wherein (a) if said βγΙΡ is fused to said RET donor, said GPCR is fused to said RET acceptor; (b) if said ΡγΙΡ is fused to said RET acceptor, said GPCR is fused to said RET donor; and (c) if said ΡγΙΡ is fused to said first fragment of said reporter protein, said GPCR is fused to said second fragment of said reporter protein.

2. The biosensor system of item 1 , wherein said Gy protein is fused to said RET donor, RET acceptor or second fragment.

3. The biosensor system of item 1 or 2, wherein said RET donor, RET acceptor or second fragment is fused at the N-terminus of said Gp or Gy protein.

4. The biosensor system of any one of items 1 to 3, wherein said RET donor, RET acceptor or first fragment is fused at the C-terminus of said βγΙΡ.

5. The biosensor system of any one of items 1 to 4, wherein said βγΙΡ is fused to said RET acceptor and said Gp protein, Gy protein or GPCR is fused to said RET donor.

6. The biosensor system of any one of items 1 to 5, wherein said RET donor is a bioluminescent protein.

7. The biosensor system of item 6, wherein said bioluminescent protein is a luciferase. 8. The biosensor system of item 7, wherein said luciferase is a Renilla luciferase.

9. The biosensor system of any one of items 1 to 8, wherein said RET acceptor is a fluorescent protein.

10. The biosensor system of item 9, wherein said fluorescent protein is a GFP.

1 1 . The biosensor system of any one of items 1 to 4, wherein said βγΙΡ is fused to said first fragment, and said Gp protein, Gy protein or GPCR is fused to said second fragment. 12. The biosensor system of item 1 1 , wherein said reporter protein is a bioluminescent protein.

13. The biosensor system of item 12, wherein said bioluminescent protein is a luciferase.

14. The biosensor system of item 13, wherein said luciferase is a Renilla luciferase.

15. The biosensor system of item 14, wherein said first fragment comprises about residues 1 to 110 of Renilla luciferase, and said second fragment comprises about residues 1 1 1 to 31 1 of Renilla luciferase.

16. The biosensor system of any one of items 1 to 15, wherein the first component further comprises a plasma membrane (P )-targeting moiety fused to said βγΙΡ or said RET donor, RET acceptor or first fragment.

17. The biosensor system of item 16, wherein said PM-targeting moiety is fused at the C- terminus of said RET donor, RET acceptor or first fragment.

18. The biosensor system of item 16 or 17, wherein said PM-targeting moiety comprises a prenylation motif.

19. The biosensor system of item 16, wherein said prenylation motif is the prenylation motif of human KRAS splice variant b.

20. The biosensor system of item 19, wherein said PM-targeting moiety comprises the amino acid sequence KKKKKKSKTKCVIM (SEQ ID NO:37).

21 . The biosensor system of any one of items 16 to 20, further comprising a flexible linker between (i) said RET donor, RET acceptor or first fragment and (ii) said PM-targeting moiety.

22. The biosensor system of item 21 , wherein said flexible linker has a length corresponding to about 50 to about 500 amino acids.

23. The biosensor system of item 22, wherein said flexible linker has a length corresponding to about 200 amino acids.

24. The biosensor system of any one of items 1 to 23, wherein said recombinant Ga protein is human Ga_q, Ga_s, Ga_n, Ga_i2, Ga_i3, Ga_t -cone, G Gdt-gust. GC⁽ _Z, Ga_oA, Ga_oB, GC⁽ ₀|f, GC⁽ii , GO-|2,

Ga₁₃, Ga₁₄, and Ga₁₅/Ga₁₆ protein, or a promiscuous or non-selective Ga variant thereof, for example a mutated Ga polypeptide comprising a mutation at a position corresponding to residue 66, 67 and/or 75 of human Ga_q protein as described herein.

25. The biosensor system of any one of items 1 to 24, wherein said βγΙΡ is GRK2 or GRK3. 26. The biosensor system of any one of items 1 to 25, wherein (i) if said second component comprises a fused Gp protein, said first and second biosensors further comprises a recombinant Gy protein, or (ii) if said second component comprises a fused Gy protein, said first and second biosensors further comprises a recombinant Gp protein.

27. The biosensor system of any one of items 1 to 25, wherein the biosensor system defined in (A) further comprises a G-protein-coupled receptor (GPCR). 28. The biosensor system of any one of items 1 to 27, wherein the biosensor system defined in (B) further comprises a recombinant Gp protein and/or a recombinant Θγ protein.

29. The biosensor system of any one of items 1 to 28, wherein said first biosensor is present in a first cell and said second biosensor is present in a second cell.

30. The biosensor system of any one of items 1 to 28, wherein in the biosensor system defined in (A), said first biosensor is present in a first membrane preparation and said second biosensor is present in a second membrane preparation.

31 . The biosensor system of any one of items 1 to 30, wherein the biosensor system defined in (A) comprises a plurality of second biosensors, wherein each of said second biosensors comprises a different recombinant Ga protein.

32. The biosensor system of item 31 , wherein said different recombinant Ga proteins are at least two of the following Ga proteins: Ga_q, Ga_s, Ga_n , Ga_i2, Ga_i3, Ga_t._COne, Ga_t._r0d, Ga_t.g_Ust, Ga_Zl Ga_oA, Ga_oB, Ga₀,_f, Go,-, , Ga ₂, Ga₁₃, Ga₁₄, and Ga₁₅/Ga₁₆.

33. A nucleic acid comprising a sequence encoding the first, second and third components defined in any one of items 1 to 26.

34. The nucleic acid of item 33, further comprising a sequence encoding a Gy protein or a Gp protein.

35. The nucleic acid of item 33 or 34, further comprising one or more translation regulatory sequences.

36. The nucleic acid of item 35, wherein said one or more translation regulatory sequences are Internal Ribosome Entry Site (IRES).

37. A biosensor for detecting G-protein activity comprising:

(i) a first component comprising a Gpy interacting protein (βγΙΡ) fused to (a) a RET donor; (b) a RET acceptor or (c) a first fragment of a reporter protein; and

(ii) a second component comprising a fused plasma membrane (PM)-targeting moiety, wherein said PM-targeting moiety is fused to (a) a RET donor; (b) a RET acceptor or (c) a second fragment of said reporter protein;

wherein (a) if said βγΙΡ is fused to said RET donor, said PM-targeting moiety is fused to said RET acceptor; (b) if said βγΙΡ is fused to said RET acceptor, said PM-targeting moiety is fused to said RET donor; and (c) if said βγΙΡ is fused to said first fragment of said reporter protein, said PM-targeting moiety is fused to said second fragment of said reporter protein.

38. The biosensor of item 37, wherein said PM targeting moiety is a PM protein or a fragment thereof that localizes to the PM .

39. The biosensor of item 38, wherein said PM protein or fragment thereof comprises (a) a palmitoylation, myristoylation, and/or prenylation signal sequence and/or (b) a polybasic sequence. 40. The biosensor of item 39, wherein said polybasic sequence and prenylation signal sequence are from human KRAS splice variant b.

41 . The biosensor of item 40, wherein said PM targeting moiety comprises the amino acid sequence KKKKKKSKTKCVI M (SEQ ID NO:37) .

42. The biosensor of any one of items 37 to 41 , wherein said biosensor further comprises a third component that comprises a recombinant Ga protein.

43. The biosensor of item 42, wherein said recombinant Ga protein is of the Gq family.

44. The biosensor of item 43, wherein said recombinant Ga protein is Ga_q or Ga-n .

45. A method for determining whether a test agent modulates the activity of a GPCR, said method comprising:

(1) providing a biosensor comprising the elements defined in (A) , (B) or (C):

(A)

(i) a first component comprising a Gpy interacting protein (βγΙΡ) fused to (a) a RET donor; (b) a RET acceptor or (c) a first fragment of a reporter protein; (ii) a second component comprising a fused Gp protein or a fused Gy protein, wherein said Gp protein or said Gy protein is fused to (a) a RET donor; (b) a RET acceptor or (c) a second fragment of said reporter protein,

wherein (a) if said βγΙΡ is fused to said RET donor, said Gp or Gy protein is fused to said RET acceptor; (b) if said βγΙΡ is fused to said RET acceptor, said Gp or Gy protein is fused to said RET donor; and (c) if said βγΙΡ is fused to said first fragment of said reporter protein, said Gp or Gy protein is fused to said second fragment of said reporter protein;

(iii) a third component comprising a recombinant Ga protein; and

(iv) a fourth component comprising said GPCR;

(B)

(i) a first component comprising a Gpy interacting protein (pylP) fused to (a) a RET donor; (b) a RET acceptor or (c) a first fragment of a reporter protein;

(ii) a second component comprising said GPCR fused at its C-terminal to (a) a RET donor; (b) a RET acceptor or (c) a second fragment of said reporter protein; (iii) a third component comprising a recombinant Ga protein;

wherein (a) if said βγΙΡ is fused to said RET donor, said GPCR is fused to said RET acceptor; (b) if said βγΙΡ is fused to said RET acceptor, said GPCR is fused to said RET donor; and (c) if said βγΙΡ is fused to said first fragment of said reporter protein, said GPCR is fused to said second fragment of said reporter protein; or (C) (i) a first component comprising a Θβγ interacting protein (βγΙΡ) fused to (a) a RET donor; (b) a RET acceptor or (c) a first fragment of a reporter protein;

(ii) a second component comprising a fused plasma membrane (P )-targeting moiety, wherein said PM-targeting moiety is fused to (a) a RET donor; (b) a RET acceptor or (c) a second fragment of said reporter protein;

wherein (a) if said βγΙ Ρ is fused to said RET donor, said PM-targeting moiety is fused to said RET acceptor; (b) if said βγΙΡ is fused to said RET acceptor, said PM-targeting moiety is fused to said RET donor; and (c) if said βγΙΡ is fused to said first fragment of said reporter protein, said PM-targeting moiety is fused to said second fragment of said reporter protein;

(iii) a third component comprising a recombinant Ga protein; and

(iv) a fourth component comprising said GPCR; and

(2) measuring the signal emitted by said RET acceptor or reporter protein in the presence and absence of said test agent;

wherein a higher signal measured in the presence of the agent is indicative that said test agent increases the activity of said GPCR, and a lower signal measured in the presence of the agent is indicative that said agent inhibits the activity of said GPCR.

46. The method of item 44, wherein said biosensors comprise one or more of the features defined in items 2 to 32 and 38 to 44.

47. A method for determining whether a Ga protein is activated by a GPCR agonist, said method comprising:

(a) measuring the signal emitted by said RET acceptor or reporter protein in the presence and absence of said GPCR agonist in the first and second biosensors of the biosensor system of any one of items 1 to 32, and

(b) identifying whether the Ga protein is activated by said GPCR agonist based on the signal emitted by said RET acceptor or reporter protein;

wherein a higher increase of the signal measured in the presence of the GPCR agonist in said second biosensor relative to said first biosensor is indicative that the Ga protein is activated by said GPCR agonist, and wherein a similar or lower increase, or a decrease, of the signal measured in the presence of the GPCR agonist in said second biosensor relative to said first biosensor is indicative that said the Ga protein is not activated by said GPCR agonist.

48. A method for determining whether a Ga protein is activated by a GPCR agonist, said method comprising:

(a) measuring the signal emitted by a RET acceptor or reporter protein in the presence and absence of said GPCR agonist in a first biosensor comprising: (i) a first component comprising a G y interacting protein (βγΙΡ) fused to (a) a RET donor; (b) a RET acceptor or (c) a first fragment of a reporter protein; and

(ii) a second component comprising a fused G-protein coupled receptor (GPCR) , wherein said GPCR is fused at its C-terminal to (a) a RET donor; (b) a RET acceptor or (c) a second fragment of said reporter protein;

(b) measuring the signal emitted by a RET acceptor or reporter protein in the presence and absence of said GPCR agonist in a second biosensor comprising:

(i) the first and second components defined in (a); and

(ii) a third component comprising a recombinant form of said Ga protein;

wherein (a) if said βγΙΡ is fused to said RET donor, said GPCR is fused to said RET acceptor; (b) if said βγΙΡ is fused to said RET acceptor, said GPCR is fused to said RET donor; and (c) if said βγΙΡ is fused to said first fragment of said reporter protein, said GPCR is fused to said second fragment of said reporter protein;

49. The method of item 47, wherein said biosensors comprise one or more of the features defined in items 38 to 44.

50. The method of item 45, further comprising

(3) measuring the signal emitted by said RET acceptor or reporter protein in

(a) the second biosensor(s) defined in element (A) of any one of items 1 to 31 ,

(b) the biosensor defined in element (B) of any one of items 1 to 31 , or (c) the biosensor of any one of items 42 to 44,

in the presence and absence of a test agent and in the presence of a GPCR agonist, wherein said recombinant Ga protein is coupled to said GPCR; and

(4) determining whether said test agent is an inhibitor of said Ga protein;

wherein a lower signal measured in the presence of the test agent is indicative that said test agent is an inhibitor of said Ga protein, and a similar or higher signal measured in the presence of the test agent is indicative that said test agent is not an inhibitor of said Ga protein.

51 . A method for determining whether a test agent is an inhibitor of a Ga protein of interest, said method comprising:

(1) contacting

(a) the second biosensor(s) defined in element (A) of any one of items 1 to 32,

(b) the biosensor defined in element (B) of any one of items 1 to 32, or

(c) the biosensor of any one of items 42 to 44; with a GPCR agonist, wherein said recombinant Ga protein corresponds to said Ga protein of interest;

(2) measuring the signal emitted by said RET acceptor or reporter protein in the presence and absence of said test agent; and

(c) determining whether said test agent is an inhibitor of said Ga protein,

wherein a lower signal measured in the presence of the test agent is indicative that said test agent is an inhibitor of said Ga protein of interest, and a similar or higher signal measured in the presence of the test agent is indicative that said test agent is not an inhibitor of said Ga protein of interest.

52. A method for determining whether a test agent is an activator of a Ga protein of interest, said method comprising:

(1) contacting

(a) the second biosensor(s) defined in element (A) of any one of items 1 to 32,

(b) the biosensor defined in element (B) of any one of items 1 to 32, or (c) the biosensor of any one of items 42 to 44;

with a GPCR antagonist, wherein said recombinant Ga protein corresponds to said Ga protein of interest;

(3) determining whether said test agent is an activator of said Ga protein,

wherein a higher signal measured in the presence of the test agent is indicative that said test agent is an activator of said Ga protein of interest, and a similar or lower signal measured in the presence of the test agent is indicative that said test agent is not an activator of said Ga protein of interest.

53. The method of any one of items 45 to 52, wherein said RET donor is a bioluminescent protein, and wherein said method further comprises contacting the biosensor with a substrate for said donor bioluminescent protein.

54. The method of item 53, wherein said substrate is a luciferin.

55. The method of item 54, wherein said luciferin is a coelenterazine.

56. The method of item 55, wherein said coelenterazine is Coelenterazine 400A.

57. The method of any one of items 45 to 56, wherein the biosensor comprises a RET donor and a RET acceptor, and wherein said method further comprises: (i) measuring signal emitted by said RET donor, and (ii) determining the ratio [RET acceptor signal / RET donor signal].

58. A mutated Ga polypeptide comprising a mutation at a position corresponding to residue 67 and/or residue 75 of human Ga_q protein.

59. The mutated Ga polypeptide of item 58, wherein said mutation is a substitution. 60. The mutated Ga polypeptide of item 58 or 59, wherein said mutation is at a position corresponding to residue 67 of human Ga_q protein.

61 . The mutated Ga polypeptide of item 60, wherein said mutation is a substitution for a non-aromatic residue.

62. The mutated Ga polypeptide of item 61 , wherein non-aromatic residue is cysteine.

63. The mutated Ga polypeptide of item 58 or 59, wherein said mutation is at a position corresponding to residue 75 of human Ga_q protein.

64. The mutated Ga polypeptide of item 63, wherein said mutation is a substitution for a non-aromatic residue.

65. The mutated Ga polypeptide of item 64, wherein said non-aromatic residue is glycine.

66. A nucleic acid comprising a sequence encoding the mutated Ga polypeptide of any one of items 58 to 65.

67. A plasmid or vector comprising the nucleic acid of item 66.

68. A cell comprising the nucleic acid of item 65 or the plasmid of item 67.

Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.

BRIEF DESCRIPTION OF DRAWINGS

In the appended drawings:

FIGs. 1A to 1 C show schematic diagrams illustrating the principle underlying the use of the βγΙΡ-based biosensor for G-protein activation, using a GPCR as an example of G-protein activator. The assay is based on the competition between the Ga subunit and the βγΙΡ for the binding to the ΰβγ dimer. While in the inactive form, the Ga subunit of the heterotrimeric G- protein is tightly bound to the ΰβγ dimer. Upon ligand binding to the receptor, the Ga subunit switches from a GDP-bound form to a GTP-bound form, resulting in its dissociation from the ΰ γ subunits, allowing βγΙΡ to be recruited to the free G$y subunits. The interaction between βγΙΡ and will thus reflect the activation of a specific G-protein, upon receptor stimulation. Different methods of detection can be used to assess this interaction between βγΙΡ and ΰ γ, such as resonance energy transfer (RET) approaches (FIG. 1A) or protein complementation (PC) assays (FIG. 1 B). In resonance energy transfer approaches, the βγΙΡ and σ ν are tagged with an energy donor and acceptor, and upon G-protein activation, an increase in RET signal is observed. In the case of protein complementation assay, the βγΙΡ and G y are fused to fragments of a fluorescent protein or luminescent enzyme, and following G-protein activation, the complementation of the two fragments will lead to an increase in the fluorescence signal or enzyme activity. FIG. 1 C shows theoretical scenarios and corresponding interpretation of results for the βγΙΡ-based biosensor of G-protein activation. Three different scenarios are depicted in FIG. 1 C using BRET as an example of detection method. In scenario 1 (left) the cells are transfected with all components of the biosensor except for the Ga subunit of the heterotrimeric G-protein. The lack of a subunits causes the excess Gpy subunits to interact with the βγΙΡ at basal state. In scenario 2 (middle) , all the components of the biosensor are transfected but the Ga subunit that is overexpressed (Ga- is not functionally coupled to the receptor of interest. Scenario 3 (right) shows a typical response of the biosensor when all its components are expressed along with the proper Ga subunit (Ga₂) for the receptor of interest. In this case, receptor activation leads to an increase in BRET signal which is caused by the recruitment of GFP-tagged βγΙΡ to the RLuc-tagged Gpy subunits previously coupled to the specific Ga subunit.

FIG. 2 presents some of the different constructs tested for optimization of the βγΙΡ- based G-protein activation biosensor. In FIG. 2A, the structure of GRK2/3 is presented. GRK2/3 arbour different functional domains, a calmodulin binding domain (CAM), an RGS (Regulator of G protein Signaling) domain that can be inactivated by the D1 10A substitution described herein, a catalytic domain for its kinase activity and that can be inactivated by the K220R substitution described herein, and a Pleckstrin homology domain (PH domain) that binds to PIP₂ and Gp subunits of heterotrimeric G-proteins. These interactions promote GRK translocation to the plasma-membrane and its activation. Phosphorylation of the C-terminal portion of GRK (serine 670 and 685) has been reported to modulate its activity. Four different GFP-tagged constructs for GRK2 and GRK3 were tested, two based on the complete GRK coding sequence and two on the C-terminal PH domain/Gp binding domain, with GFP at either the N-terminal or the C-terminal portion of GRK. Both Gp and Gy subunits were tested as a fusion with a BRET tag and can be used to monitor GRK/Gpy interaction.

FIG. 2B and 2C show the testing of different ratios (titration) of the four different GRK constructs (FIG. 2A) and responses obtained for p-iAR activation of Ga₁₅ (FIG. 2B) and in for thromboxane A2 receptor (TPaR)-mediated activation of Ga-n (FIG. 2C) . Titrations of BRET donor to acceptor were performed on HEK293 cells transfected with constructs encoding a receptor and a Ga

in FIG 2B & TPaR/Ga-n in FIG. 2C), Gp1 , RIUCII-GY5 (0.5 ng per well of a 96-well plate) and variable amount of GRK2 constructs tagged with GFP10 (up to 75ng/well) . The cells were treated with vehicle or agonist (1 μΜ isoproterenol and 100 nM U- 46619 for cells expressing p-iAR and TPaR, respectively) for 15 min. The BRET ratios were reported in function of GFP-construct expression (evaluated in fluorescence) over Rlucl l construct expression (evaluated in bioluminescence). These results indicate that the full length GRK tagged at its C-terminal with the BRET donnor (GFP) is giving the best dynamic window in term of amplitude of BRET signal and stability of response over a wider range of ratios donnor to acceptor. FIGs. 3A to 3C show the G-protein activation profile of TPaR using a βγΙΡ-based biosensor. FIG. 3A: HEK293 cells transiently expressing the TPaR along with GRK2-GFP, f?luc- Gy5, Gp1 and the indicated Ga, were exposed to 100nM of U-46619 or vehicle for 15 min, prior to BRET measurements. The mock condition is without any Ga subunit overexpression. FIG. 3B: BRET values obtained for the agonist treated cells in FIG. 3A expressed as a percentage of the BRET values obtained with the corresponding cells treated with vehicle. Mock condition is used to determine the threshold of a positive response. FIG. 3C: Dose-response curves using the agonist U-46619 for Ga_q, Ga-|₃, Gai₄, Gai₅, Ga_qG66K and Ga_qY67C activation of the TPaR using GRK2-GFP/Rluc-Gy5/Gpi biosensor.

FIGs. 4A to 4J show the G-protein activation profiles for the dopamine D2 receptor

(D₂R), the α -adrenergic receptor (a_1BAR) , and the a_2c-adrenergic receptor (a_2cAR) using a βγΙΡ-based biosensor. HEK293 cells transiently expressing the D₂R (FIGs. 4A and 4B) , a _BAR (FIGs. 4C and 4D) or a_2CAR (FIGs. 4E and 4F) along with GRK2-GFP, Rluc-Gy5, Gpi and the indicated Ga, were stimulated with the following agonists, rotigotine (FIGs. 4A and 4B), phenylepinephrine (FIGs. 4C, 4D, 4E) or epinephrine (FIG. 4E) for 15min prior to BRET measurements. FIGs. 4A, 4C and 4E: Data are expressed as a percentage of the BRET signal obtained in vehicle-treated cells. Mock condition without any Ga subunit overexpression, was used to determine the threshold of a positive response. As presented in FIGs. 4A, 4C and 4E, G-protein with promiscuous activation properties such as Ga_qY67C could be used to monitor receptor activation (see position and surrounding sequence at FIG.14). These promiscuous mutants of Ga, could be used as positive controls for receptor activation which, could be useful for characterizing antagonists or screening for orphan receptor agonists. FIG. 4B Dose- response curves for Rotigotine, a D2R agonist, with selected Ga proteins (Ga_n and four promiscuous Ga_q mutants: G66K, G66D, Y67C and F75G) using the GRK2-GFP/Rluc-Gy5/Gpi biosensor. In FIG 4D, dose-response curves are presented for phenylephrine, an a-adrenergic agonist, with a _BAR and selected Ga proteins (Ga-π and Ga_q) using the GRK2-GFP/Rluc- Gy5/Gp1 biosensor. In FIG 4E, dose-response curves of Ga_z activation were obtained for different adrenergic agonists: epinephine, norepinephrine, phenylephrine and isoproterenol, from HEK293 cells expressing a_2CAR, Ga_z, GRK2-GFP, Rluc-Gy5 and Gpi . FIG. 4F shows the G-protein activation profile for a_2CAR using two different a_2CAR agonists, epinephrine and phenylephrine. These results show that a βγΙΡ-based biosensor can be used to establish G- protein activation and pharmacological profiles of different receptors and ligands. In FIG. 4G to 4J, dose-response curves for epinephrine/a₂cAR-promoted Ga_z activation were obtained with different combinations of Gpy subunits. HEK293 cells were transfected with constructs encoding the a_2CAR, Ga_z, GRK2-GFP, different Rluc-tagged Gy (FIG. 4G and 4H) and a Gp (Gpi in FIG. 4G and the short variant of Gp3 (Gp3sh) , in FIG. 4H). In FIGs. 4I and 4J, cells were transfected with constructs encoding the a₂cAR, Ga_z, GRK2-GFP, Rluc-tagged Gy (Gy1 in FIG. 4I and Gy5, in FIG. 4J) and different Gp. These results show that combinations of both Gp and Gy subunits can lead to distinct pharmacological profile of G-protein activation. These differences could be, in part, linked to distinct pharmalogical profiles observed with different cells and tissues expressing not only a specific set of Ga subunits but also different combination and levels of Gp and Gy subunits. These results show that a ΡγΙΡ-based biosensor could be useful to study and better understand these differences.

FIGs. 5A to 5D show that the ΡγΙΡ-based biosensor can be used to characterize and validate G-protein modulators selectivity and mode of action. FIGs. 5A and 5B show the selective inhibition of Ga_n by PTX (a Gai/Ga₀ blocker) , and Ga_q by Ubo-Qic (an analog of the Ga_q inhibitor: YM-254890). HEK293 cells expressing the TPaR and Ga_q (FIG. 5A) or the D₂R and Ga_n (FIG. 5B) , along with Gpi , Rluc-Gy5 and GRK2-GFP, were pre-treated with PTX, Ubo- Qic or vehicle (control) and then exposed to increasing concentrations of U-46619 (FIG. 5A) or rotigotine (FIG. 5B) for 15 min, before recording BRET signals. In FIG. 5C, TPaR-mediated G- protein activation was used to validate Ubo-Qic inhibitor selectivity. Cells co-expressing TPaR and the biosensor GRK2-GFP/Rluc-Gy5/Gp1 + the indicated Ga subunit were pretreated with Ubo-Qic and exposed to either vehicle or an agonist: U-46619 (100nM). These results show that, from the Ga_q family (Ga_q, Gan , Ga-|₄ and Ga-|₅), only Ga-|₅ is insensitive to Ubo-Qic. The Ga-i2 and Ga-|₃ proteins are also insensitive to Ubo-Qic. FIG. 5D, the ΡγΙΡ-based biosensor was used to reveal the Ubo-Qic sensitivity of mutant Ga_q activation. Ga_q substitutions were introduced at position 67 (see FIG. 14) . Only the substitutions of this tyrosine residue that are resistant to Ubo-Qic inhibition (Y67C, Y67G , Y67S & Y67L) also showed promiscuous properties, indicating that this residue could also be important for controlling G-protein activation. The substitution of the Phe75 residue to glycine led to only a partial Ubo-Qic mediated inhibition of activation (FIG. 5D) and also to a promiscuous phenotype (see FIG. 4A) .

FIGs. 6A and 6B show the kinetics of the pylP-based G-protein activation biosensor responses upon receptor activation. FIG. 6A: HEK293 cells transiently expressing the D₂R along with Ga^ , Gpi , Rluc-Gy5 and GRK2-GFP were exposed to 1 μΜ of rotigotine or vehicle while BRET measurements were performed at regular intervals. FIG. 6B: HEK293 cells transiently expressing the TPaR along with Gan , Gp1 , Rluc-Gy5 and GRK2-GFP were exposed to 100 nM of U-46619 or vehicle while BRET measurements were performed at regular intervals. In both cases, the agonist and vehicle were added to the cells after 30 sec of measurements.

FIGs. 7A and 7B show the Z' factor evaluation for the ΡγΙΡ-based G-protein activation biosensor. HEK293 cells transiently expressing the D₂R and Gan (FIG. 7A) or the TPaR and Ga-,-, (FIG. 7B), along with Gpi , Rluc-Gy5 and GRK2-GFP were exposed to either 1 μΜ of rotigotine (FIG. 7A), 100 nM of U-46619 (FIG. 7B) or vehicle (FIGs. 7A, 7B) for 15 min. BRET ratios are represented for each individual well of a 96-well plate. Z' factor, for these representative experiments, were evaluated at 0.79 and 0.89 for D₂R (FIG. 7A) and TPaR (FIG. 7B), respectively.

FIGs. 8A to 8C show a ligand profiling with the βγΙΡ-based G-protein activation biosensor. FIG. 8A: G-protein activation profile of HEK293 cells transiently expressing the angiotensin II type 1 receptor (AT1 R) along with Gp1 , Rluc-Gy5, GRK2-GFP and the indicated Ga, stimulated with 1 μΜ angiotensin II for 15 min prior to BRET measurements. FIG. 8B: G- protein activation profiles for a saturating concentration of angiotensin II analogs (1 μ ) for Ga_q, Ga-ι-ι and Ga₁₂. Results in FIGs. 8A and 8B are expressed as a percentage of the BRET signal obtained in vehicle treated cells, and mock condition without any Ga subunit overexpression, was used to determine the threshold of a positive response. FIG. 8C: Dose-response curves obtained using the Angll and DVG ligands for Ga_q and Ga₁₂ activation of the AT1 R using the GRK2-GFP/f?luc-Gy5/G(31 biosensor. Data is expressed as the % of the Angll response obtained for each G-protein.

FIGs. 9A and 9B show the use of a protein complementation-based detection method to assess G-protein activation with the βγΙΡ-based biosensor; an Rluc protein complementation assay (Rluc-PCA). FIG. 9A: Z' factor obtained for HEK293 cells transfected with the TPaR, GRK2-f?lucF1 , RlucF2-Gy5, Gpi and Go,i subunit, stimulated with 100 nM of U-46619 or vehicle for 10 min. Luminescence values are represented for each individual well of a 96-well plate. Z' factor, for this representative experiment, was evaluated at 0.53. FIG. 9B: Dose- response curves using the agonist U-46619 for Ga-n activation of the TPaR using GRK2- RlucF1/RlucF2-Gy5/Gpi biosensor.

FIGs. 10A to 10C show the use of GRK3 as a βγΙΡ to assess G-protein activation. FIG. 10A: Dose response curves obtained from HE 293 cells transiently expressing the D₂R along with Ga_n , Gp1 , Rluc-Gy5 and GR 2-GFP (black circles) or GR 3-GFP (white triangles), exposed to increasing concentrations of the agonist rotigotine for 15 min prior to BRET measurements. FIG. 10B: Kinetics of GRK3-based biosensor response for HEK293 cells transfected with D₂R, Ga_n, Gpi , Rluc-Gy5 and GRK3-GFP, exposed to 1 μΜ of rotigotine or vehicle while BRET measurements were performed at regular interval. The agonist and vehicle were injected to the cells after 30 sec of measurements. FIG. 10C: Z' factor evaluation of GRK3- based biosensor for HEK293 cells transfected with D₂R, Ga_n, Gpi , Rluc-Gy5 and GRK3-GFP, exposed to either 1 μΜ of rotigotine or vehicle for 15 min. BRET ratios are represented for each individual well of a 96-well plate. Z' factor, for this representative experiment, was evaluated at 0.71 . FIGs. 11 A to 11 D show the results of experiments performed using a polycistronic vector encoding a βγΙΡ-based G-protein activation biosensor. FIG. 11 A: Schematic diagram illustrating the polycistronic construct which encodes the following proteins: GRK2-GFP, Rluc- Gj5 and Gp1 . A G-protein activation profile is presented in FIG. 11 B, for HEK293 cells co- transfected with constructs encoding for TPaR, a Ga (either Ga_q, Ga-n , Ga₁₂, Ga₁₃, Ga₁₄ or Ga, 5 161 Mock condition was without Ga) and a polycistronic construct (described in FIG. 11 A) encoding the GFP-tagged WT GRK2 or a RGS-dead mutant (D1 10A) of GRK2. TPaR activation by its agonist (100nM of U46619) led to similar results and profile with both polycistronic constructs, indicating that a functional RGS domain is not a prerequisite for GRK2 recruitment. FIG. 11C: Dose-response curves using the agonist U-46619 for Gan activation of the TPaR using the polycistronic construct (with WT GRK2) described in FIG. 11 A. In FIG. 11 D, a Z' factor was obtained for HEK293 cells transfected as in FIG. 11 C, and stimulated with 100 nM of U- 46619 or vehicle for 15 min. BRET ratios are represented for each individual well of a 96-well plate. Z' factor, for this representative experiment, was evaluated at 0.80.

FIGs. 12A and 12B show a membrane-anchored βγΙΡ-based G-protein activation biosensor. FIG. 12A: Schematic diagram illustrating the principle underlying the use of the membrane anchored GRK2 (GRK2-mem)-based biosensor and the associated DNA construct encoding GRK2-GFP-mem. FIG. 12B: Membrane preparations were obtained from HEK293 cells transfected with TPaR, Gp1 , RIUCI I-GY5, GRK2-GFP or GRK2-GFP-mem, in absence or presence of Gan , which were stimulated with 100 nM of U-46619 or vehicle for 15 min. BRET experiments were then performed on those membrane preparations. Data are expressed as a percentage of the BRET signal obtained in vehicle treated cells.

FIGs. 13A to 13C show that substitutions reported for affecting GRK2 functions (RGS and catalytic) or its regulation by phosphorylation, do not prevent nor significantly promote its recruitment to activated G-proteins. In FIG. 13A, HEK293 cells co-expressing TPaR, Ga_q, Gp1 , RIUCI I-GY5 and variants of GRK2-GFP (WT= solid square, RGS-dead D1 10A mutant = empty circle, and catalytically-dead 220R mutant = empty triangle) were stimulated with increasing doses of U46619. As shown in FIG. 11 B and 13A, a functional RGS domain is not required (nor does it promote) the Ga_q response detected with the biosensor. A catalytically-dead mutant of GRK2 can also be used with this biosensor (FIGs. 13A and 13C) in two configurations: G- protein activation measured as an increase in BRET from GR 2-GFP interaction with free GP1 /RIUCII-GY5 (FIG. 13A) and from Rlucll-GRK2 interaction with free Gpl /GFP10-Gy5 (FIG. 13C). Using these mutants would minimize the side effects of overexpressing a functional kinase, which is known to inhibit Ga_q-mediated activation of PLC through its RGS domain. The use of such mutants could be advantageous for applications that require monitoring of multiple signalling pathways through multiplexing of sensors or of different assays. In FIG. 13B, HEK293 cells were transfected as in FIG. 13A but with either WT GRK2 (solid square) or mutants that would prevent (S670A = open triangles, S676A = empty diamonds and S685A = empty circles) or mimic (S670D = solid triangles, S676D = solid diamonds and S685D = solid circles) phosphorylation of its C-terminal binding domain. Phosphorylation of GRK2 on these serine residues by ERK, PKA and CDK2-CyclinA, is known to modulate its activity (Cong et a/. , The Journal of Biological Chemistry, 276, 15192-15199; Pitcher ef a/. , The Journal of Biological Chemistry, 274, 34531 -34534; Penela et a/., PNAS, 107(3): 1 1 18-1 123; Choudhary ef a/. , Mol Cell. 2009 36(2): 326-39) . However, the results presented in FIG. 13B provide evidence that GRK2 recruitment to Gpy could be insensitive to regulation by different signalling events. I n FIG. 13C, HEK293 cells co-expressing TPaR, Ga_q, Gpi , GFP-Gy5 and variants of Rlucl l-GRK2 (VVT = solid squares, and catalytically-dead 220R mutant = empty triangles) were stimulated with increasing doses of U46619. Both configurations of BRET donor and acceptor with tags at either N-terminus (FIG. 13C) or C-terminus of GRK2 (FIG. 13A) led to similar results, providing evidence that the configuration of the biosensor is flexible.

FIG. 14 shows a sequence alignment of human G-protein a subunits (SEQ ID NOs: 1 - 17) and substitutions leading to promiscuous coupling properties. The human Ga subunits of heterotrimeric G-proteins were aligned using DIALIGN tool (http://bibiserv.techfak.uni- bielefeld.de/dialign/submission.html), formatted using the Boxshade tool (http://www.ch.embnet.org/software/BOX_form.html) and a region centered on Linkerl is presented. The residues that show high conservation throughout the Ga subunits are identified with a black and grey background. The Linkerl and a helices from secondary structure prediction are also identified.

FIG. 15A shows a schematic diagram illustrating a biosensor comprising a βγΙΡ (GRK) tagged with a RET acceptor (A) and a GPCR tagged at its C-terminal with a RET donor (D). The assay is also based on the competition between the Ga subunit and the βγΙΡ for the binding to the ΰβγ dimer, which is bound to the C-terminal portion of the GPCR. While in the inactive form, the Ga subunit of the heterotrimeric G-protein is tightly bound to the ΰβγ dimer. Upon ligand binding to the GPCR, the Ga dissociates from the ΰβγ subunits, allowing βγΙΡ to be recruited to the free ββγ subunits and bringing the RET acceptor in close proximity to the RET donor linked to the GPCR, thus inducing/increasing the BRET signal. FIGs. 15B and 15C show dose- response curves for G-protein activation, obtained with the biosensor described in FIG. 15A. HEK293 cells co-expressing TPaR-Rlucl l, different Ga (Ga_q = solid square, Ga^ = solid triangle, Ga₁₄ = solid diamond and Ga₁₂ = empty circle), ΰβ1 , Gy5 and, either the WT GRK2- GFP (FIG. 15B) or the mutant D1 10A GRK2-GFP (FIG. 15C), were stimulated with increasing doses of U46619. The dose-response curves show similar profiles in FIGs. 15B and 15C indicating, as in FIGs. 11 B and 13A but with a different biosensor configuration, that a functional RGS is not required to recruit a βγΙΡ to an activated G-protein. FIG. 16A shows a schematic diagram illustrating a biosensor comprising a βγΙΡ (GRK) tagged with the RET donor (D) and a plasma-membrane marker: a RET acceptor (A) tagged with a plasma-membrane targeting and anchoring sequence (e.g. , a CAAX domain). The assay is also based on the competition between the Ga subunit and the βγΙΡ for the binding to the G y dimer, at the plasma-membrane. While in the inactive form, the Ga subunit of the heterotrimeric G-protein is tightly bound to the Gpy dimer. Upon ligand binding to the GPCR, the Ga dissociates from the G$y subunits, allowing βγΙΡ to be recruited to the free G y subunits, at the plasma-membrane which, leads to an increase in density of RET donor (βγΙΡ-D) and acceptor (plasma-membrane marker, A-CAAX), thus inducing/increasing the BRET signal. FIG. 16B shows dose-response curves for G-protein activation, obtained with the biosensor described in FIG. 16A. HEK293 cells co-expressing TPaR, different Ga (Ga_q = solid square, Ga-n = solid triangle, Mock condition (no Ga) = empty circle), G , Gy5, Rlucl l-GRK2 and rGFP-CAAX, were stimulated with increasing doses of U46619. Dose-response curves in FIG. 16B are similar to those obtained in FIGs. 3C, 9B and 11 C with different configuration of biosensors. In FIG. 16C, a Z' factor was obtained for HEK293 cells transfected as in FIG. 16B, and stimulated with 100 nM of U-46619 or vehicle for 15 min. BRET ratios are represented for each individual well of a 96-well plate. Z' factor, for this representative experiment, was evaluated at 0.89.

FIG. 17A shows the amino acid sequence of human GRK2 (SEQ I D NO: 18), with positions D1 10, K220R, S670, S676 and S685 (mutated in some of the constructs described herein) in bold, the putative PH domain underlined, and the C-terminal portion thereof (GRK2 Cterm, SEQ ID NO:50) used in some of the constructs described herein in italics.

FIG. 17B shows the amino acid sequence of human GRK3 (SEQ ID NO: 19) with the putative PH domain underlined, and the amino acid sequence of the C-terminal portion thereof (GRK3 Cterm, SEQ ID NO:51 ) used in some of the constructs described herein in italics.

FIG. 17C shows the amino acid sequence of PLE HG2 (SEQ ID NO:20) with the putative PH domain underlined.

FIG. 17D shows the amino acid sequence of GFP10 (SEQ ID NO:38) used in the experiments described herein.

FIG. 17E shows the amino acid sequence of Renilla reniformis GFP (rGFP, SEQ ID NO:46) used in the experiments described herein.

FIG. 17F shows the amino acid sequence of RLucl l (SEQ ID NO:39) used in the experiments described herein.

DISCLOSURE OF INVENTION

Terms and symbols of genetics, molecular biology, biochemistry and nucleic acids used herein follow those of standard treatises and texts in the field, e.g. Kornberg and Baker, DNA Replication, Second Edition (W.H. Freeman, New York, 1992); Lehninger, Biochemistry, Second Edition (Worth Publishers, New York, 1975); Strachan and Read, Human Molecular Genetics, Second Edition (Wiley-Liss, New York, 1999); Eckstein, editor, Oligonucleotides and Analogs: A Practical Approach (Oxford University Press, New York, 1991); Gait, editor, Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, 1984); and the like. All terms are to be understood with their typical meanings established in the relevant art.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element. Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

In the studies described herein, the present inventors have shown that a βγΙΡ- competition-based biosensor may be used to monitor G-protein activation, without the need to modify the receptor and/or the Ga subunits. As it is based on competition, a single biosensor is needed to study all the different G-proteins and establish G-protein activation/coupling profiles based on the co-transfected Ga subunit. G-protein activation profiles are not only important for characterizing receptors and drug targets, but may also be useful in the drug discovery process for identifying, characterizing and optimizing GPCRs ligands with biased signaling properties associated with therapeutic efficacy and reduced side effects.

The present disclosure relates to a universal biosensor for monitoring G-protein activation, without having to modify either Ga protein subunits or G-protein activators (such as G-protein-coupled receptors (GPCR), activators of G-protein signalling (AGS), regulators of G- protein signalling or other chemical and biological entities). More specifically, the disclosure relates to the use of a G y-interacting protein (βγΙΡ) to monitor the activation of the various hetero-trimeric G-proteins. Advantageously, the signalling biosensor disclosed herein allows for a sensitive and quantitative assay which can be used in large-scale screening assays and structure-activity relationship studies for the identification of ligands (agonists, antagonists, inverse agonists, allosteric modulators, etc.) targeting G-protein activity. Additionally, the biosensor disclosed herein represents a tool for assessing G-protein activation profiles and allows for compound profiling by addressing which specific G-proteins are activated upon stimulation.

As shown in FIG. 1 , the system according to an embodiment of the present disclosure is based on the competition between the Ga subunit and the βγΙΡ for the binding to the Gpy dimer. While in the inactive form, the Ga subunit of the hetero-trimeric G-protein is tightly bound to the ΰ γ dimer. Upon ligand binding to the receptor, the Ga subunit switches from a GDP- bound form to a GTP-bound form, resulting in its dissociation from the ββγ subunits, allowing βγΙΡ to be recruited to the free Θβγ subunits. The interaction between βγΙΡ and Θβγ will thus reflect the activation of a specific G-protein, upon receptor stimulation.

The present inventors have also shown that it is possible to monitor G-protein activation using a biosensor that measures the recruitment/localization of a βγΙΡ (e.g., GRK), tagged with a BRET donor (e.g., RLuc) , at the plasma membrane (where it interacts with the G y complex bound to the GPCR) using a plasma membrane-targeting moiety tagged with a complementary BRET acceptor (e.g., rGFP). The increase in the concentration/density of βγΙΡ at the plasma membrane, an indirect measure of the recruitment of the βγΙΡ to the Gpy complex, is detected by an increase in the BRET signal.

The present inventors have further shown that it is possible to monitor G-protein activation using a biosensor that measures the recruitment of a βγΙΡ (e.g., GRK), tagged with a BRET donor (e.g., RLuc), to a GPCR-tagged with a complementary BRET acceptor (e.g., rGFP) (FIG. 15).

In this context, the present disclosure relates to a βγΙΡ-based G-protein activation biosensor and a system using such biosensor to assess activation of specific G-proteins promoted by their activators. The system comprises a G-protein activator; a Ga protein; and the biosensor described herein. The present disclosure further relates to a method for detecting G- proteins activation using the system disclosed herein.

The present disclosure thus relates to a biosensor system for detecting G-protein activity, said biosensor system comprising the elements defined in (A) or (B):

(A) (i) a first biosensor comprising: a first component comprising a G y interacting protein (βγΙΡ) fused to (a) a RET donor; (b) a RET acceptor or (c) a first fragment of a reporter protein; and a second component comprising a fused G protein or a fused Gy protein, wherein said G protein or said Gy protein is fused to (a) a RET donor; (b) a RET acceptor or (c) a second fragment of said reporter protein; (ii) a second biosensor comprising: the first and second components defined in (i); and a third component comprising a recombinant Ga protein; wherein

(a) if said βγΙΡ is fused to said RET donor, said G$ or Gy protein is fused to said RET acceptor;

(b) if said βγΙΡ is fused to said RET acceptor, said ΰβ or Gy protein is fused to said RET donor; and (c) if said βγΙΡ is fused to said first fragment of said reporter protein, said G$ or Gy protein is fused to said second fragment of said reporter protein; or

(B) (i) a biosensor comprising a first component comprising a G y interacting protein (βγΙΡ) fused to (a) a RET donor; (b) a RET acceptor or (c) a first fragment of a reporter protein; a second component comprising a fused G-protein coupled receptor (GPCR), wherein said GPCR is fused at its C-terminal to (a) a RET donor; (b) a RET acceptor or (c) a second fragment of said reporter protein; a third component comprising a recombinant Ga protein; wherein (a) if said βγΙΡ is fused to said RET donor, said GPCR is fused to said RET acceptor; (b) if said βγΙΡ is fused to said RET acceptor, said GPCR is fused to said RET donor; and (c) if said βγΙΡ is fused to said first fragment of said reporter protein, said GPCR is fused to said second fragment of said reporter protein.

The present disclosure thus relates to a biosensor comprising: (1 ) a first component comprising a GPy-interacting protein (βγΙΡ) fused to (a) a RET donor; (b) a RET acceptor or (c) a first fragment of a reporter protein; (2) a second component comprising a fused Gp protein or a fused Gy protein, wherein said Gp protein or said Gy protein is fused to (a) a RET donor; (b) a RET acceptor or (c) a second fragment of said reporter protein; (3) a third component comprising a recombinant Ga protein, wherein said recombinant Ga protein is a promiscuous or non-selective Ga protein, for example a Ga protein comprising a mutations at a position corresponding to residue 66, 67 and/or 75 of human Ga_q, as described herein. In an embodiment, the biosensor further comprises a GPCR (native or recombinant), preferably an orphan GPCR.

In an embodiment, the biosensor defined above further comprises a recombinant Gp protein and/or a recombinant Gy protein. In a further embodiment, the biosensor defined above further comprises a recombinant Gp protein and a recombinant G protein. In an embodiment, the biosensor defined above further comprises a GPCR, in a further embodiment a recombinant GPCR.

In another aspect, the present disclosure thus relates to a biosensor comprising (i) a first component comprising a Gpy interacting protein (ΡγΙΡ) fused to (a) a RET donor; (b) a RET acceptor or (c) a first fragment of a reporter protein; and (ii) a second component comprising a fused plasma membrane (P )-targeting moiety, wherein said PM-targeting moiety is fused to (a) a RET donor; (b) a RET acceptor or (c) a second fragment of said reporter protein; wherein (a) if said βγΙΡ is fused to said RET donor, said PM-targeting moiety is fused to said RET acceptor; (b) if said ΡγΙΡ is fused to said RET acceptor, said PM-targeting moiety is fused to said RET donor; and (c) if said βγΙΡ is fused to said first fragment of said reporter protein, said PM-targeting moiety is fused to said second fragment of said reporter protein.

In one non-limiting embodiment, activity of the herein described biosensor is detectable based on a technique selected from resonance energy transfer (RET) such as bioluminescence resonance energy transfer (BRET) or fluorescence resonance energy transfer (FRET) ; protein complementation assay or protein-fragment complement assay (PCA) such as enzyme fragment complementation (EFC) or bimolecular fluorescence complementation (BiFC); and the like (see FIG. 1). Such techniques are known in the art and employ tags/moieties which may be fused at the C-terminal, the N-terminal or within the protein elements of the biosensor.

In resonance energy transfer approaches, the ΡγΙΡ and Gpy are tagged with an energy donor and acceptor, and upon G-protein activation, an increase in RET signal is observed. In the case of protein complementation assay, the βγΙΡ and Gpv are tagged with fragments of a reporter protein, such as a fluorescent protein or luminescent enzyme, and following G-protein activation, the complementation of the two fragments will lead to an increase in the reporter protein signal, for example the fluorescence signal or enzyme activity.

Resonance energy transfer (abbreviated RET) is a mechanism describing energy transfer between two chromophores, having overlapping emission/absorption spectra. When the two chromophores (the "donor" and the "acceptor"), are within a short distance (e.g., 10-100 Angstroms) of one another and their transition dipoles are appropriately oriented, the donor chromophore is able to transfer its excited-state energy to the acceptor chromophore through non-radiative dipole-dipole coupling. One type of RET is Bioluminescence Resonance Energy Transfer (BRET) that is based on the non-radiative transfer of energy between a donor bioluminophore (bioluminescent enzyme such as luciferase) and an acceptor fluorophore (ex: GFP or YFP). Another type of RET is Fluorescence Resonance Energy Transfer (FRET) involves the transfer of energy from an excited donor fluorophore to an adjacent acceptor fluorophore. For example, CFP and YFP, two color variants of GFP, can be used as donor and acceptor, respectively.

As used herein, the term "fluorescent protein" refers to any protein that becomes fluorescent upon excitation at an appropriate wavelength. A broad range of fluorescent proteins have been developed that feature fluorescence emission spectral profiles spanning almost the entire visible light spectrum. Non-limiting examples of green Fluorescent Protein include EGFP, GFP10, Emerald, Superfolder GFP, Azami Green, mWasabi, TagGFP, TurboGFP, AcGFP, ZsGreen and T-Sapphire. Non-limiting Examples of blue fluorescent protein include EBFP, EBFP2, Azurite and mTagBFP. Non-limiting examples of Cyan Fluorescent proteins include ECFP, mECFP, Cerulean, mTurquoise, CyPet, AmCyan l , Midori-lshi Cyan, TagCFP, mTFP1 (Teal). Non-limiting examples of Yellow fluorescent proteins include EYFP, Topaz, Venus, mVenus, mCitrine, mAmetrine, YPet, TagYFP, PhiYFP, ZsYellowl and mBanana. Non-limiting Examples of orange fluorescent proteins include Kusabira Orange, Kusabira Orange2, mOrange, mOrange2, dTomato, dTomato-Tandem, TagRFP, DsRed, DsRed2, DsRed-Express (T1 ), DsRed- onomer and mTangerine. Non-limiting Examples of red fluorescent proteins include mRuby, mApple, mStrawberry, AsRed2, mRFP1 , JRed, mCherry, HcRed l , mRaspberry, dKeima-Tandem, HcRed-Tandem, mPlum and AQ143.

"Overlap" as used in the context of the present invention refers to the ability of the emitted light from a donor fluorescent protein or a luminescent enzyme (e.g. , luciferase) to be of a wavelength capable of excitation of a fluorophore (acceptor fluorescent protein) placed in close proximity, usually within about 10-100 A (about 1 -10 nm) . Accordingly, the donor fluorescent or luminescent protein and the acceptor fluorescent protein are selected so as to enable the transfer of energy from the donor fluorescent or luminescent protein, attached to a first component of the biosensor, to the acceptor fluorescent protein attached to a second component of the biosensor, when the first and second components are in close proximity (i.e., in the form of a complex or in the same cellular compartment, such as the plasma membrane). Such transfer of energy is commonly referred to as "Fluorescence (or Forster) Resonance Energy Transfer" or "FRET" (if the donor protein is a fluorescent protein), or "Bioluminescence Resonance Energy Transfer" or "BRET" (if the donor protein is a bioluminescent protein). Thus, any combination of donor fluorescent or luminescent protein and acceptor fluorescent proteins may be used in accordance with the present invention as long as the above criteria are met. Such combinations are typically referred as FRET or BRET pairs. The choice of a suitable fluorophore for use in a BRET assay will be known to one of skill in the art. In one embodiment, fluorophores include green fluorescent protein - wild type (GFP-wt), yellow fluorescent protein (YFP), Venus, Topaz, ZsYellowl , mOrange2, mKeima, blue fluorescent protein (BFP), cyan fluorescent protein (CFP), Tsapphire, mAmetrine, green fluorescent protein-2 (GFP2), renilla GFP (rGFP) and green fluorescent protein- 10 (GFP10), or variants thereof. Fluorescent proteins having an excitation peak close to 400 nm may be particularly suitable. More particular examples of fluorophores include mAmetrine, cyan fluorescent protein (CFP), and GFP10. Representative examples of FRET pairs include BFP/CFP, BFP/GFP, BFP/YFP, BFP/DsRed, CFP/GFP, CFP/YFP, CFP/mVenus, GFP/YFP, GFP2/YFP, GFP/DsRed, TagBFP/TagGFP2, TagGFP2/TagRFP and the like (see, e.g., Muller et a/., Front. Plant Sci., 4: 413, 2013). Representative examples of BRET pairs include luciferase (Luc)/GFP, Luc/Venus, Luc/Topaz, Luc/GFP-10, Luc/GFP-2, Luc/YFP, Luc/rGFP, and the like.

As used herein, the term "luciferase" refers to the class of oxidative enzymes used in bioluminescence and which is distinct from a photoprotein. One example is the firefly luciferase (EC 1 .13.12.7) from the firefly Photinus pyralis (P. pyralis luciferase). Several recombinant luciferases from several other species including luciferase from Renilla reniformis (GENBANK: AAA29804) and variants thereof (e.g., a stable variant of Renilla Luciferase e.g., Rlucll (GENBANK : AAV52877.1), Rluc8 (GENBANK: EF446136.1) Gaussia Luciferase (Glue, GENBANK: AAG54095.1), NanoLuc® Luciferase (Promega®) are also commercially available. Any luciferase can be used in accordance with the present invention as long as it can metabolize a luciferase substrate such as luciferins. Luciferins are a class of light-emitting heterocyclic compounds that are oxidized in the presence of luciferase to produce oxyluciferin and energy in the form of light. Non-limiting examples of luciferins include D-luciferin, imidazopyrazinone-based compounds such as coelenterazine (coelenterazine 400A (DeepBlueC™), coelenterazine H and e-coelenterazine derivatives such as methoxy e- Coelenterazine (Prolume® Purple I from NanoLight Technology®), ViviRen™ (from Promega®), Latia luciferin ((£)-2-methyl-4-(2,6,6-trimethyl-1 -cyclohex-1 -yl)-1 -buten-1 -ol formate), bacterial luciferin, Dinoflagellate luciferin, etc. Luciferase substrates may have slightly different emission spectra and will thus be selected to favor the optimal energy transfer to the acceptor. In an embodiment, the luciferase is wild-type (or native) Renilla Luciferase. In an embodiment, the luciferase is the stable variant of Renilla luciferase Rluc8. In another embodiment, the luciferase is Gaussia luciferase (GLuc). In a specific embodiment, the luciferase is Renilla Luciferase II (Rlucll) and the luciferin is coelenterazine 400A.

In an embodiment, one of the following BRET configurations is used in the biosensors and methods described herein: BRET1 that comprises coelenterazine-h (coel-h) and a YFP (YFP) or a GFP from Renilla (rGFP); BRET2 that comprises coelenterazine-400a (coel-400a) and a UV-excited (uvGFP) or a GFP from Renilla (rGFP); or BRET3 that comprises coel-h or v- coelenterazine (from Nanolight Technology®) and the monomeric orange FP (mOrange). In a further embodiment, RLucll is used in the above-noted BRET configurations. In another embodiment, one of the following BRET configurations is used in the biosensors and methods described herein: Rlucll/coel-400a/enhanced blue (EB) FP2, Rlucll/coel-400a/super cyan fluorescent protein (SCFP3A), Rlucll/coel-400a/mAmetrine or Rlucll/coel-400a/GFP10. In an embodiment, the BRET donor is a Renilla luciferase (e.g., RLucll) and the BRET acceptor is a Renilla GFP (e.g., Renilla reniformis GFP).

In PCA, each of the proteins (e.g., βγΙΡ and Gp/Gy, or GPCR) is covalently linked to incomplete fragments of a reporter protein, and the interaction between βγΙΡ and Gp/Gy brings the fragments of the reporter protein in close enough proximity to allow them to form a functional reporter protein whose activity can be measured. Any protein that can be split into two parts and reconstituted non-covalently may be used in the PCA-based biosensor. The term "reporter protein" refers to a protein that can be detected (e.g., by fluorescence, spectroscopy, luminometry, etc.) easily and that is not present normally (endogenously) in the system used. Typical reporter proteins used in PCA include enzymes (whose activity may be measured using a suitable substrate) such as dihydrofolate reductase (DHFR), β-lactamase, β-galactosidase or proteins that give colorimetric or fluorescent signals such as a luciferase (e.g., Renilla luciferase), GFP and variants thereof.

In another non-limiting embodiment, the RET or PCA tags are located on: (i) the βγΙΡ and the Gp protein, or (ii) the βγΙΡ and the Gy protein. In a further non-limiting embodiment, the βγΙΡ and the Gfi or Gy subunits are tagged at their N-terminus, C-terminus or at any internal region within the proteins. In one embodiment, the βγΙΡ and the Gfi or Gy subunits are tagged at their N-terminus or C-terminus. In one non-limiting embodiment, the herein described PCA tags added to the βγΙΡ and the G$ or Gy subunits can be, without being limited to, a fluorophore, a luciferase or a fragment thereof comprising a portion of a fluorescent protein or luminescent enzyme.

"GPCR" refers to full length native GPCR molecules as well as mutant/variant GPCR molecules. A list of GPCRs is given in Foord et al (2005) Pharmacol Rev. 57, 279-288, which is incorporated herein by reference, and an updated list of GPCRs is available in the IUPHAR-DB database (Harmar AJ, et al. (2009) IUPHAR-DB: the IUPHAR database of G protein-coupled receptors and ion channels. Nucl. Acids Res. 37 (Database issue): D680-D685; Sharman JL, et al. , (2013) IUPHAR-DB: updated database content and new features. Nucl. Acids Res. 41 (Database Issue): D1083-8; Alexander SPH, Benson HE, Faccenda E, Pawson AJ, Sharman JL, Spedding , Peters JA and Harmar AJ, CGTP Collaborators. (2013) The Concise Guide to PHARMACOLOGY 2013/14: G Protein-Coupled Receptors. Br J Pharmacol. 170: 1459-1581). In an embodiment, the GPCR is an orphan GPCR. The term "orphan GPCR" as used herein refers to an apparent receptor that has a similar structure to other identified GPCRs but whose endogenous ligand has not yet been identified. GPCR orphan receptors are often given the name "GPR" followed by a number, for example GPR1 . An updated list of orphan GPCRs is available in the IUPHAR-DB database described above.

In an embodiment, the GPCR is fused at its C-terminal to a RET donor or RET acceptor, in a further embodiment a RET donor, such as a luciferase (RLuc).

The term "recombinant" as used herein refers to a protein molecule which is expressed from a recombinant nucleic acid molecule, i.e. a nucleic acid prepared by means of molecular biology/genetic engineering techniques, for example a protein that is expressed following transfection/transduction of a cell (or its progeny) with a nucleic acid (e.g., present in a vector) encoding the protein (as opposed to a protein that is naturally expressed by a cell).

The term variant (or mutant) as used herein refers to a protein which is substantially similar in structure (amino acid sequence) and biological activity to the corresponding native protein. It includes fragments comprising one or more domains of a native protein, as well as fusion proteins comprising the native protein or a fragment thereof. A variant may comprises one or more mutations (substitutions, deletions, insertions) relative to the native protein in order to generate a protein having certain desired features, for example being constitutively active, inactive, altered binding to one or more ligands, etc. Individual substitutions, deletions or additions that alter, add or delete a single amino acid or-nucleotide or a small percentage of amino acids or nucleotides in the sequence create a "conservatively modified variant," where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants and alleles of the invention.

"Homology" or "identity" and "homologous" or "identical" refer to sequence and/or structural similarity between two polypeptides or two nucleic acid molecules. Homology/identity can be determined by comparing each position in the aligned sequences. A degree of homology/identity between nucleic acid or between amino acid sequences is a function of the number of identical or matching nucleotides or amino acids at positions shared by the sequences. As the term is used herein, a nucleic acid sequence is homologous to another sequence if the two sequences are substantially identical and the functional activity of the sequences is conserved (as used herein, the term 'homologous' does not infer evolutionary relatedness). Two nucleic acid sequences are considered substantially identical if, when optimally aligned (with gaps permitted), they share at least about 50% sequence similarity or identity, or if the sequences share defined functional motifs. In alternative embodiments, sequence similarity in optimally aligned substantially identical sequences may be at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%. As used herein, a given percentage of homology/identity between sequences denotes the degree of sequence identity in optimally aligned sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity, though preferably less than about 25 % identity, with any of the sequences described herein.

In one non-limiting embodiment, the system includes a living cell, a membrane preparation, or both. The system defined herein is, but not limited to, a membrane preparation and said βγΙΡ is tethered to the membrane via a membrane targeting linker, for example a protein/peptide linker comprising a plasma membrane (P )-targeting domain (e.g., a plasma membrane-anchoring signal peptide). This plasma membrane-targeting domain may be, without being limited thereto, a lipid group covalently bound to the peptide chain such as palmitoylation, myristoylation or prenylation modifications (as the membrane anchoring signal from KRAS for example (Hancock 2003)), a transmembrane domain, or a polybasic region (as the one present in GRK5 for instance).

In an embodiment, the PM-targeting moiety comprises a CAAX motif (C is cysteine residue, AA are two aliphatic residues, and X represents any amino acid. CAAX motifs are found in "CAAX proteins" that are defined as a group of proteins with a specific amino acid sequence at C-terminal that directs their post translational modification. CAAX proteins encompass a wide variety of molecules that include nuclear lamins (intermediate filaments) such as prelamin A, lamin B1 and lamin B2, Ras and a multitude of GTP-binding proteins (G proteins) such as Ras, Rho, Rac, and Cdc42, several protein kinases and phosphatases, etc. (see, e.g., Gao et al., Am J Transl Res. 2009; 1 (3): 312-325). The proteins that have a CAAX motif or box at the end of the C-terminus typically need a prenylation process before the proteins migrate to the plasma membrane or nuclear membrane and exert different functions. In an embodiment, the CAAX box is derived from a human RAS family protein, for example HRAS, NRAS, Ral-A, KRAS4A or KRAS4B. The last C-terminal residues of RAS, NRAS, KRAS4A or KRAS4b (referred to as the hypervariable region or HVR) are depicted below, with the putative minimal plasma membrane targeting region in italics and the CAAX box underlined (see, e.g., Ahearn et al., Nature Reviews Molecular Cell Biology 13: 39-51 , January 2012): HRAS: KLNPPDESGPGCMSCKCJ/LS; (SEQ ID NO:40); NRAS: KLNSSDDGTQGCMGLPCW ; (SEQ ID NO: 41); KRAS4A: KISKEEKTPGCWC/ KC// ; (SEQ ID NO:42); KRAS4B: KM SKDG KKKKKKSKTKC VIM: (SEQ ID NO:43); Ral-A/Ral1 : KNGKKKRKSLAKRIRERCCIL (SEQ ID NO:44). In an embodiment, the membrane targeting moiety comprises the last 4 residues of the sequences depicted above. In a further embodiment, the membrane targeting moiety comprises the last 10 residues of the sequences depicted above. In an embodiment, the membrane targeting moiety comprises the C-terminal portion (e.g., about the last 10-30 or 15- 25 amino acids) of a CAAX protein, for example a human RAS family protein, e.g., about the last 10-30, 15-25 or 20 amino acids of a human RAS family protein.

In an embodiment, the PM-targeting moiety comprises the sequence KKKKKKSKTKCVIM (SEQ ID NO: 37) from KRAS4B. In another embodiment, the PM targeting moiety comprises the the plasma-membrane targeting palmitoylation sequence from hRas and prenylation signal sequence from Ral-A/Ral1 (sequence: CMSCKCCIL, SEQ ID NO:45).

Several proteins also contain a non-lipid, polybasic domain that targets the PM such as Ras small GTPases, phosphatase PTEN, nonreceptor tyrosine kinase Src, actin regulators WASP and MARCKS, and G protein-coupled receptor kinases (GRKs) such as GRK5. In an embodiment, the polybasic domain is from GRK5, and comprises the sequence SPKKGLLQRLFKRQHQNNSKS (SEQ ID NO:46). In an embodiment, the PM-targeting moiety is fused at the C-terminal end of a RET donor or acceptor, and in a further embodiment a RET acceptor such as a GFP (e.g., rGFP). In another embodiment, the PM-targeting moiety is fused at the C-terminal end of a RET donor or acceptor, and in a further embodiment a RET acceptor such as a GFP (e.g., rGFP), and the RET donor or acceptor is fused at its N-terminal to a βγΙΡ, such as a GRK protein or a G y-interacting fragment/variant thereof.

According to the present disclosure, G-protein activator include, but is not limited to, classical activation of G-proteins by GPCRs and other proteins that can also modulate the activity of these hetero-trimeric G-proteins, such as regulators of G-protein signalling (RGS), activators of G-protein signalling (AGS), and resistance to inhibitors of cholinesterase 8 proteins (Ric-8). In some of these non-canonical signalling pathways, the guanine exchange factor (GEF) activity classically exerted by GPCRs is replaced by another protein such as Ric-8 for example (Boularan and Kehrl, 2014).

In one embodiment, the G-protein activator is a member of the GPCR family.

Ga protein subunit as defined herein includes, but is not limited to, the 17 different known isoforms, their splice variants, and any mutated Ga proteins, for example those leading to non-selective/promiscuous Ga. In one non-limiting embodiment, the herein described Ga protein is selected amongst any of the natural mammalian Ga proteins, which includes Ga_q, Ga_s, Ga_n , Ga_i2, Ga_i3, Ga_t._cone, Ga_t._rod, Ga_t._gust, Ga_z, Ga_oA, Ga_oB, Ga₀,_f, Gan , Ga₁₂, Ga₁₃, Ga₁₄, and Ga-i5 i₆ (now designated GNA15), the splice variants of these isoforms, as well as functional variants thereof. In an embodiment, the Ga protein subunit is of the G, family. In an embodiment, the Ga protein subunit is of the G_s family. In an embodiment, the Ga protein subunit is of the G_q family. In an embodiment, the Ga protein subunit is of the G ₂/13 family. In an embodiment, the Ga protein is a promiscuous or non-selective Ga protein. In a further embodiment, the Ga protein is a mutated Ga proteins (e.g. , Ga_q proteins) having a substitution at any of the following positions, G66, Y67, F75 and any combinations thereof, or equivalent conserved substitution in other Ga subtypes, which results in non-selective Ga proteins that are activated by any GPCRs), including orphan receptors (i.e. that are able to interact with GPCRs independently from the preferential natural coupling of these receptors to specific Ga proteins, also commonly referred to as "promiscuous" Ga proteins), are also included in the present disclosure. In an embodiment, the recombinant Ga protein used in the biosensors/methods described herein is a promiscuous Ga protein, and the GPCR is an orphan GPCR.

In another aspect, the present disclosure relates to a mutated Ga polypeptide comprising a mutation at a position corresponding to residue 67 and/or residue 75 of human Ga_q protein. Said mutation may be an insertion, deletion, or a substitution, for example a non- conservative substitution. FIG. 14 discloses an alignment of the sequences of representative hGa proteins that may be mutated according to the present invention, with the positions corresponding 67 and 75 of Ga_q indicated by arrows. The skilled person would understand that depending on the number of residues N-terminal of the positions corresponding 67 and 75 of Ga_q in a particular Ga, the numbering of the residue varies. For example, in hGa₁₄, the residue corresponding to position 67 of Ga_q is residue 63 (Y). Similarly, in hGa-|₂, the residue corresponding to position 67 of Ga_q is residue 85 (F). Thus, the present invention encompasses for example a mutated Ga₁₄ polypeptide comprising a mutation at position 63 (e.g., a substitution for a non-aromatic residue) , and a mutated Ga-|₂ polypeptide comprising a mutation at position 85 (e.g. , a substitution for a non-aromatic residue), which correspond to a mutation at position 67 of Ga_q. Any mutated Ga polypeptide comprising a mutation at one or more of positions corresponding to residue 67 and/or residue 75 of human Ga_q protein are encompassed by the present disclosure.

In an embodiment, the present invention relates to a mutated Ga polypeptide comprising any one of the sequences set forth in SEQ ID NOs: 1 - 17, wherein the residue corresponding to residue 67 and/or residue 75 of human Ga_q protein is mutated. In an embodiment, the mutation is at a position corresponding to residue 67 of human Ga_q protein. In an embodiment, the mutation is at a position corresponding to residue 67 and is a substitution for a non-aromatic residue, in a further embodiment cysteine. In another embodiment, the mutation is at a position corresponding to residue 75 of human Ga_q protein, and is a substitution for a non-aromatic residue, in a further embodiment the non-aromatic residue is glycine. Such mutated Ga polypeptide may be used in any of the biosensors and/or methods described herein. In one non-limiting embodiment, the mutated Ga_q protein comprises one of the following substitutions, Ga_qG66K, Ga_qY67C and Ga_qF75G, resulting in non-selective Ga proteins.

In another aspect, the present disclosure relates to a nucleic acid comprising a sequence encoding the above-defined mutated Ga polypeptide. In another aspect, the present disclosure relates to a plasmid or vector comprising the above-defined nucleic acid. In another aspect, the present disclosure relates to a cell (host cell) comprising the above-defined nucleic acid or vector. In another aspect, the present invention provides a kit comprising a nucleic acid encoding the mutated Ga polypeptide defined herein. In an embodiment, the cell has been transfected or transformed with a nucleic acid encoding the mutated Ga polypeptide defined herein. The invention further provides a recombinant expression system, vectors and cells, such as those described above, for the expression of the mutated Ga polypeptide defined herein, using for example culture media and reagents well known in the art. The cell may be any cell capable of expressing mutated Ga polypeptide defined above. Suitable host cells and methods for expression of proteins are well known in the art. Any cell capable of expressing the mutated Ga polypeptide defined above may be used. For example, eukaryotic host cells such as mammalian cells may be used (e.g., rodent cells such as mouse, rat and hamster cell lines, human cells/cell lines). In another embodiment, the above-mentioned cell is a human cell line, for example an embryonic kidney cell line (e.g., HEK293 or HEK293T cells).

In embodiments, the herein described Gp protein is selected amongst any of the known Gp proteins, which includes Gpi , Gp2, Gp3 (e.g. , a short variant of Gp3, Gp3sh), Gp4 and Gp5 (Gp5-S or Gp5-L), the splice variants of these isoforms, and functional variants thereof. In a further embodiment, the Gp protein is Gpi . In another embodiment, the Gp protein is Gp3. In a further embodiment, the Gp protein (e.g. , Gp1) is N-terminally tagged with a BRET acceptor, such as a GFP.

In embodiments, the herein described Gy protein is selected amongst any of the known human Gy proteins, which include Gy^ , Gy2, Gy3, Gy4, Gy5, Gy7, Gy8, Gy9, Gy10, Gy1 1 , Gy12 and Gy13, and functional variants thereof. In a further embodiment, the Gy protein is Gy5. In a further embodiment, the Gy protein (e.g. , Gy5) is N-terminally tagged with a BRET donor, such as a luciferase. In another embodiment, the Gy protein (e.g. , Gy5) is N-terminally tagged with a BRET acceptor, such as a GFP. In another embodiment, the Gy protein (e.g., Gy5) is N- terminally tagged with a first domain of a PCA-compatible reporter protein, e.g. a luciferase (e.g. , Renilla luciferase).

In an embodiment, the herein described ΡγΙΡ is a protein that interacts with Gpy dimer upon dissociation of the Gapy heterotrimer and that comprises a pleckstrin homology (PH) domain, such as a G-protein coupled receptor kinase (GRK) protein (GRK2 or GRK3) or functional fragment thereof that comprises the C-terminal pleckstrin homology (PH) domain of a GRK protein (i.e. that maintain the ability to interact with a Θβγ dimer), a pleckstrin homology domain containing family G (with RhoGef domain) member 2 (PLEKHG2). The amino acid sequences of GRK2, GRK3 and PLEKHG2 are depicted in FIGs. 17A-C, with the PH domain underlined. In one non-limiting embodiment, the herein described GRK protein (GRK2 or GRK3) or fragment thereof that maintains the ability to interact with a ΰβγ dimer (e.g., that comprises the C-terminal pleckstrin homology (PH) domain of the GRK, such as a C-terminal fragment comprising the sequence set forth in SEQ ID NO:50 or 51 ) is C-terminally tagged with a BRET acceptor, such as a fluorophore. In an embodiment, the βγΙΡ is GRK2 or GRK3 or a variant/fragment thereof, and it is C-terminally fused with a BRET acceptor, such as a GFP. In another embodiment, the βγΙΡ is a variant of a GRK protein that comprises a mutation that inactivates its regulator of G protein signaling (RGS) domain ("RGS-dead" variant) . In a further embodiment, the "RGS-dead" variant of a GRK protein comprises a mutation at a position corresponding to residue D1 10 of GRK2, for example a D to A substitution. The RGS domain of native human GRK2 (UniProtKB accession P25098) and GRK3 (UniProtKB accession P35626) spans about residues 54 to 175. In another embodiment, the βγΙΡ is a variant of a GRK protein that comprises a mutation that inactivates its kinase domain ("kinase-dead" variant) . In a further embodiment, the "kinase-dead" variant of a GRK protein comprises a mutation (e.g., non- conservative substitution) at a position corresponding to residue K220 of GRK2, for example a K to D substitution. The kinase domain of GRK2 (UniProtKB accession P25098) and GRK3 (UniProtKB accession P35626) spans about residues 191 to 453. In another embodiment, the βγΙΡ is a variant of a GRK protein that comprises a mutation in its C-terminal domain, e.g. , within the last 30 C-terminal residues. In a further embodiment, the mutation is a serine residue located within the C-terminal domain, and more particularly a serine that may be phosphorylated in the native protein. In a further embodiment, the mutation (e.g., non-conservative substitution) is at a position corresponding to residue S670, S676 and/or S685 of GRK2, for example an S to A and/or an S to D substitution.

In embodiments, the domains of the fusion molecules described herein may be covalently linked either directly (e.g., through a peptide bond) or "indirectly" via a suitable linker moiety, e.g., a linker of one or more amino acids or another type of chemical linker (e.g. , a carbohydrate linker, a lipid linker, a fatty acid linker, a polyether linker, PEG , etc. In an embodiment, one or more additional domain(s) may be inserted before (N-terminal), between or after (C-terminal) the domains defined above. In an embodiment, the domains of the fusion molecules are covalently linked through a peptide bond. In another embodiment, one or more of the components of the fusion molecules are linked through a peptide linker. Linkers may be employed to provide the desired conformation of the BRET/FRET label chromophores within the labeled compound, e.g., including the separation between chromophores in a BRET/FRET pair. The linkers may be bound to the C-terminal, the N-terminal, or at an intermediate position. In one embodiment, the linkers are peptide linkers, typically ranging from 2 to 30 amino acids in length, for example about 5 to about 20-25 amino acids. The composition and length of each of the linkers may be chosen depending on various properties desired such as flexibility and aqueous solubility. For instance, the peptide linker may comprise relatively small amino acid residues, including, but not limited to, glycine; small amino acid residues may reduce the steric bulk and increase the flexibility of the peptide linker. The peptide linker may also comprise polar amino acids, including, but not limited to, serine. Polar amino acid residues may increase the aqueous solubility of the peptide linker. Furthermore, programs such as Globplot 2.3 (Linding et al. , GlobPlot: exploring protein sequences for globularity and disorder, Nucleic Acid Res 2003 - Vol. 31 , No.13, 3701 -8), may be used to help determine the degree of disorder and globularity, thus also their degree of flexibility. In an embodiment, the peptide linker comprises one or more of the amino acid sequences disclosed in the Examples below.

In one non-limiting embodiment, as illustrated in FIG. 12A, the herein described recombinant βγΙΡ-based construct comprises a βγΙΡ tagged with a fluorophore, a luciferase or a fragment thereof comprising a portion of a fluorescent protein or luminescent enzyme, a linker, preferably a flexible polypeptide linker, and a plasma membrane (P )-anchoring/targeting domain or signal for tethering the βγΙΡ to the membrane. In an embodiment, the flexible linker has a length corresponding to the length of a random amino acid sequence of about 50 to about 1000, 900, 800, 700, 600 or 500 amino acids, for example a length of about 100 to about 500, 400 or 300 amino acids, preferably a length of about 200 to 400, 200 to 300, or about 200 amino acids. In a further embodiment, the flexible linker comprises a random amino acid sequence of about 50 to about 1000, 900, 800, 700, 600 or 500 amino acids, for example a length of about 100 to about 500, 400 or 300 amino acids, preferably a length of about 200 to 400, 200 to 300, or 200 amino acids. Methods for designing flexible amino acid linkers, and more specifically linkers with minimal globularity and maximal disorder, are known in the art. Tis may be achieved, for example, using the Globplot program described above. The sequence may be further optimized to eliminate putative aggregation hotspots, localization domains, and/or interaction and phosphorylation motifs. In an embodiment, the flexible linker is located between the BRET donor or acceptor (e.g., Rluc or GFP) and the plasma membrane targeting domain. In a further embodiment, the construct has the following configuration: βγΙΡ (e.g. , GRK2) - BRET acceptor (e.g. , GFP) - flexible linker - PM targeting domain (e.g. , CAAX domain) .

In one embodiment, the present disclosure relates to a system comprising: a GPCR; a Ga protein selected from the following: Ga_q, Ga_s, Ga_n , Ga_i2, Ga_i3, Ga_t._Cone, Ga_t._rod, Gat-gust, Ga_z, Ga_oA, Ga_oB, Ga₀|_f, Ga-π , Ga₁₂, Ga₁₃, Ga-|₄, and Ga _5/16, and mutated non-selective Ga proteins as described herein; a signaling biosensor comprising a GRK protein (GRK2 or GRK3) or fragment thereof that comprises the C-terminal pleckstrin homology (PH) domain of the GRK, tagged with a fluorophore, a luciferase or a fragment thereof comprising a portion of a fluorescent protein or luminescent enzyme, a Gp protein and a Gy protein, wherein the Gp protein or the Gy protein is tagged with a fluorophore, a luciferase or a fragment thereof comprising a portion of a fluorescent protein or luminescent enzyme.

In one embodiment, the present disclosure relates to a system comprising: a GPCR; a

Ga protein selected from the following: Ga_q, Ga_s, Ga_n , Ga_i2, Ga_i3, Ga_t._cone, Ga_t._r0d, Ga_t.g_USt, Ga_z, Ga_oA, Ga₀B, Ga₀|_f, Ga-n , Ga-|₂, Ga ₃, Ga ₄, and Ga_15/16, and mutated Ga protein having a substitution at a position corresponding to any of the positions of Ga_q: G66, Y67 and/or F75; a signaling biosensor comprising a GRK protein (GRK2 or GRK3) or fragment thereof that comprises the C-terminal pleckstrin homology (PH) domain of the GRK, tagged with a fluorophore, a luciferase or a fragment thereof comprising a portion of a fluorescent protein or luminescent enzyme, a Gpi protein and a Gy5 protein, wherein the Gp protein or the Gy protein is tagged with a fluorophore, a luciferase or a fragment thereof comprising a portion of a fluorescent protein or luminescent enzyme.

In accordance with another broad non-limiting aspect, the present disclosure relates to a system for characterizing a signaling signature of a ligand, the system comprising: an activator of G-protein activity; a Ga protein; and a biosensor or system as described herein.

The present disclosure also relates to a system comprising nucleic acid sequences, which could be but is not limited to, a DNA molecule, RNA molecule, virus or plasmid, encoding proteins as defined in the present disclosure. In an embodiment, the present disclosure also relates to a nucleic acid comprising a sequence encoding one or more of the protein components (e.g. , fusion proteins) of the biosensors described herein. In an embodiment, the nucleic acid comprises a sequence encoding a (i) a βγΙΡ, (ii) a first fluorophore, a bioluminescent protein or a fragment thereof comprising a portion of a fluorescent protein or bioluminescent protein; (iii) a Gy protein; (iv) a second fluorophore, a bioluminescent protein or a fragment thereof comprising a portion of a fluorescent protein or bioluminescent protein and (v) a Gp protein. In a further embodiment, the nucleic acid further comprises one or more sequences encoding one or more linkers located between the components of the biosensor. In a further embodiment, the nucleic acid further comprises one or more transcriptional regulatory sequence(s), such as promoters, enhancers and/or other regulatory sequences, and/or one or more sequences involved in translation regulation, for example internal ribosome entry site (IRES) sequence(s).

In an embodiment, the nucleic acid is present in a vector/plasmid, in a further embodiment an expression vector/plasmid. Such vectors comprise a nucleic acid sequence capable of encoding the above-defined components (e.g., fusion proteins) of the biosensor described herein operably linked to one or more transcriptional regulatory sequence(s). The term "vector" refers to a nucleic acid molecule, which is capable of transporting another nucleic acid to which it has been linked. One type of preferred vector is an episome, i.e. , a nucleic acid capable of extra-chromosomal replication. Preferred vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors". A recombinant expression vector of the present invention can be constructed by standard techniques known to one of ordinary skill in the art and found, for example, in Sambrook et al. (1989) in Molecular Cloning: A Laboratory Manual. A variety of strategies are available for ligating fragments of DNA, the choice of which depends on the nature of the termini of the DNA fragments and can be readily determined by persons skilled in the art. The vectors of the present invention may also contain other sequence elements to facilitate vector propagation and selection in bacteria and host cells. In addition, the vectors of the present invention may comprise a sequence of nucleotides for one or more restriction endonuclease sites. Coding sequences, such as for selectable markers and reporter genes, are well known to persons skilled in the art.

A recombinant expression vector comprising a nucleic acid sequence of the present invention may be introduced into a cell (a host cell), which may include a living cell capable of expressing the protein coding region from the defined recombinant expression vector. The living cell may include both a cultured cell and a cell within a living organism. Accordingly, the invention also provides host cells containing the recombinant expression vectors of the invention. The terms "cell", "host cell" and "recombinant host cell" are used interchangeably herein. Such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

Vector DNA can be introduced into cells via conventional transformation or transfection techniques. The terms "transformation" and "transfection" refer to techniques for introducing foreign nucleic acid into a host cell, including calcium phosphate or calcium chloride co- precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, microinjection and viral-mediated transfection. Suitable methods for transforming or transfecting host cells can for example be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2^nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory manuals. "Transcriptional regulatory sequence/element" is a generic term that refers to DNA sequences, such as initiation and termination signals, enhancers, and promoters, splicing signals, polyadenylation signals which induce or control transcription of protein coding sequences with which they are operably linked. A first nucleic acid sequence is "operably-linked" with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably-linked to a coding sequence if the promoter affects the transcription or expression of the coding sequences. Generally, operably- linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in reading frame. However, since for example enhancers generally function when separated from the promoters by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably-linked but not contiguous.

In an embodiment and as depicted in FIG. 11 A, the nucleic acid or vector encodes more than one of the components (fusion proteins) of the biosensors described herein (i.e. polycistronic construct). In an embodiment, the polycistronic construct (e.g., DNA, vector) comprises a nucleic acid sequence encoding a βγΙΡ and a Gy protein, each tagged with a suitable fluorophore, a luciferase or a fragment thereof comprising a portion of a fluorescent protein or luminescent enzyme, in addition to a Gp protein. The system of the invention can be reproduced by co-transfecting this polycistronic construct with a DNA molecule comprising a nucleic acid sequence encoding a Ga protein subunit and a G-protein activator of interest.

In another aspect, the present invention provides a kit comprising the nucleic acids and/or vectors defined herein.

In another aspect, the present disclosure also provides a cell (e.g. , host cell) comprising or expressing any of the protein components (e.g. , fusion proteins, recombinant proteins) of any of the biosensors described herein. In an embodiment, the cell has been transfected or transformed with a nucleic acid encoding the mutated Ga polypeptide defined herein. The invention further provides a recombinant expression system, vectors and cells, such as those described above, for the expression of the mutated Ga polypeptide defined herein, using for example culture media and reagents well known in the art. The cell may be any cell capable of expressing mutated Ga polypeptide defined above. Suitable host cells and methods for expression of proteins are well known in the art. Any cell capable of expressing the mutated Ga polypeptide defined above may be used. For example, eukaryotic host cells such as mammalian cells may be used (e.g., rodent cells such as mouse, rat and hamster cell lines, human cells/cell lines). In another embodiment, the above-mentioned cell is a human cell line, for example an embryonic kidney cell line (e.g., HEK293 or HEK293T cells). In another aspect, the present disclosure also provides a membrane preparation comprising or expressing any of the protein components (e.g. , fusion proteins, recombinant proteins) of any of the biosensors described herein, in a further embodiment a membrane-anchored fusion protein.

The present disclosure further relates to a method for assessing a modulation in the recruitment of a GPy-interacting protein (βγΙΡ) to a G y subunit between a first condition and a second condition, said method comprising: providing one of the biosensor defined herein; measuring the BRET acceptor signal in said first and second conditions; wherein a difference in the BRET signal between said first and second conditions is indicative of a modulation in the recruitment of a GPy-interacting protein (βγΙΡ) to a Θβγ subunit between the first condition and the second condition. In an embodiment, the first condition is the presence of a test agent and the second condition is the absence of a test agent, wherein a difference in the BRET signal is indicative that the test agent modulates (increases or decreases) the recruitment of the Gpy- interacting protein (βγΙΡ) to the Gpy subunit. The recruitment of the G y-interacting protein (βγΙΡ) to the ΰβγ subunit may be used as a readout for GPCR and/or G-protein activation.

The present disclosure further relates to a method for detecting G-protein activation comprising a system described herein, the method comprising: 1 ) contacting said system with a compound that activates a G-protein, and 2) detecting the activation of the G-protein by measuring the signal of the biosensor. The method may further comprise the steps of 3) deriving G-protein functional coupling information from of the signal of the signalling biosensor, and 4) processing the information to determine the G-protein activation profile of the G-protein activator and the signalling signature of the compound. Using a biosensor system that comprises a plurality of biosensors, wherein each of the biosensors comprises a different recombinant Ga protein, it is possible to determine the G-protein coupling profile of any GPCR and/or GPCR ligand, as exemplified in FIG. 3A and 3B.

The term "compound", "agent", "test compound" or "test agent" refers to any molecule (e.g. , drug candidates) that may be screened by the method/biosensor of the invention may be obtained from any number of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means.

The present disclosure further relates to a method for determining whether a test agent modulates the activity of a GPCR, said method comprising measuring the signal emitted by a RET acceptor or reporter protein in the presence and absence of said test agent in one of the biosensor described herein; wherein a higher signal measured in the presence of the agent is indicative that said test agent increases the activity of said GPCR, and a lower signal measured in the presence of the agent is indicative that said agent inhibits the activity of said GPCR. In an embodiment, the method comprises:

(1 ) providing a biosensor comprising the elements defined in (A), (B) or (C):

(A) (i) a first component comprising a ΰ γ interacting protein (βγΙΡ) fused to (a) a RET donor; (b) a RET acceptor or (c) a first fragment of a reporter protein; (ii) a second component comprising a fused ΰβ protein or a fused Gy protein, wherein said ΰβ protein or said Gy protein is fused to (a) a RET donor; (b) a RET acceptor or (c) a second fragment of said reporter protein, wherein (a) if said βγΙΡ is fused to said RET donor, said Gp or Gy protein is fused to said RET acceptor; (b) if said βγΙΡ is fused to said RET acceptor, said Gp or Gj protein is fused to said RET donor; and (c) if said βγΙΡ is fused to said first fragment of said reporter protein, said Gp or Gy protein is fused to said second fragment of said reporter protein; (iii) a third component comprising a recombinant Ga protein; and (iv) a fourth component comprising said GPCR;

(B) (i) a first component comprising a Gpy interacting protein (βγΙΡ) fused to (a) a RET donor;

(b) a RET acceptor or (c) a first fragment of a reporter protein; (ii) a second component comprising said GPCR fused at its C-terminal to (a) a RET donor; (b) a RET acceptor or (c) a second fragment of said reporter protein; (iii) a third component comprising a recombinant Ga protein; wherein (a) if said βγΙΡ is fused to said RET donor, said GPCR is fused to said RET acceptor; (b) if said βγΙΡ is fused to said RET acceptor, said GPCR is fused to said RET donor; and (c) if said βγΙΡ is fused to said first fragment of said reporter protein, said GPCR is fused to said second fragment of said reporter protein; or

(C) (i) a first component comprising a G$y interacting protein (βγΙΡ) fused to (a) a RET donor;

(b) a RET acceptor or (c) a first fragment of a reporter protein;

(ii) a second component comprising a fused plasma membrane (P )-targeting moiety, wherein said PM-targeting moiety is fused to (a) a RET donor; (b) a RET acceptor or (c) a second fragment of said reporter protein; wherein (a) if said βγΙΡ is fused to said RET donor, said PM- targeting moiety is fused to said RET acceptor; (b) if said βγΙΡ is fused to said RET acceptor, said PM-targeting moiety is fused to said RET donor; and (c) if said βγΙΡ is fused to said first fragment of said reporter protein, said PM-targeting moiety is fused to said second fragment of said reporter protein;

(iii) a third component comprising a recombinant Ga protein; and

(iv) a fourth component comprising said GPCR; and

(2) measuring the signal emitted by said RET acceptor or reporter protein in the presence and absence of said test agent; wherein a higher signal measured in the presence of the agent is indicative that said test agent increases the activity of said GPCR, and a lower signal measured in the presence of the agent is indicative that said agent inhibits the activity of said GPCR.

In an embodiment, the above-mentioned method further comprises:

(3) measuring the signal emitted by said RET acceptor or reporter protein in the biosensor(s) defined herein in the presence and absence of a test agent and in the presence of a GPCR agonist, wherein the recombinant Ga protein is coupled to the GPCR (i.e. is known to be coupled or activated by the GPCR); and (4) determining whether said test agent is an inhibitor of said Ga protein; wherein a lower signal measured in the presence of the test agent is indicative that the test agent is an inhibitor of the Ga protein, and a similar or higher signal measured in the presence of the test agent is indicative that the test agent is not an inhibitor of the Ga protein.

In an embodiment, the term "higher signal" or "lower signal" as used herein refers to signal that is at least 10, 20, 30, 40, 45 or 50% higher (or lower) relative to the reference signal measured in the absence of the test agent. In another embodiment, the "higher signal" or "lower signal" is determined by showing a statistically significant difference (determined using a suitable statistical analysis) in the signal measured in the presence relative to the absence of the test agent, for example by combining the results obtained in a plurality of samples. Statistical analysis (ANOVA, Student i-test, Chi square, etc.) to determine significant differences between different sets of data are known in the art, and such analysis may be performed using suitable computer programs.

The present disclosure further relates to a method for identifying the Ga protein(s) activated by a GPCR agonist (G-protein profiling/signature of the agonist), said method comprising (i) measuring the signal emitted by said RET acceptor or reporter protein in the presence and absence of said GPCR agonist in a plurality of biosensors as defined herein, wherein each of the biosensors comprises a different recombinant Ga protein; (ii) identifying the Ga protein(s) activated by said GPCR agonist; wherein a higher increase of the signal measured in the presence of the GPCR agonist in a biosensor comprising a recombinant Ga protein relative to a corresponding biosensor not expressing the recombinant Ga protein is indicative that the Ga protein is activated by said GPCR agonist, and wherein a similar or lower increase, or a decrease, of the signal measured in the presence of the GPCR agonist in a biosensor comprising a recombinant Ga protein relative to a corresponding biosensor not expressing the recombinant Ga protein is indicative that the Ga protein is not activated by said GPCR agonist. In an embodiment, the method comprises: (a) measuring the signal emitted by said RET acceptor or reporter protein in the presence and absence of said GPCR agonist in the first and in the plurality of second biosensors of the biosensor system defined herein, and (b) identifying the Ga protein(s) activated by said GPCR agonist; wherein a higher increase of the signal measured in the presence of the GPCR agonist in said second biosensor relative to said first biosensor is indicative that the Ga protein is activated by said GPCR agonist, and wherein a similar or lower increase, or a decrease, of the signal measured in the presence of the GPCR agonist in said second biosensor relative to said first biosensor is indicative that said the Ga protein is not activated by said GPCR agonist.

Positive controls and negative controls may be used in the methods/assays described herein. Control and test samples may be performed multiple times to obtain statistically significant results. In an embodiment, the above-mentioned methods are high-throughput methods (high- throughput screening, HTS). The term "high-throughput screening" (HTS) as used herein refers to a method that allow screening rapidly and in parallel large numbers of compounds (hundreds, thousands) for binding activity or biological activity against target molecules. Such HTS methods are typically performed in microtiter plates having several wells, for example 384, 1536, or 3456 wells. For HTS, it is important that the readout signal be detected with high sensitivity, accuracy and reproducibility.

Methods and devices to measure the BRET signal are well known in the art. The BRET signal may be measured, for example, by determining the intensity of the BRET acceptor signal (light intensity), and/or by calculating the ratio of the signal or light intensity emitted by the BRET acceptor over the signal or light intensity emitted by the BRET donor (BRET ratio). The BRET signal may be measured using a microplate reader or microscope with a suitable filter set for detecting the BRET donor and/or BRET acceptor light emissions.

It should be understood that any combination/sub-combination of the features or embodiments described herein may be present or used in the biosensors, systems and/or methods described herein.

In an embodiment, the biosensors, systems and/or methods described herein comprises one or more of the constructs/fusion proteins and/or recombinant proteins described in the Examples below and attached Figures, for example Rluc-Gy1 to Gy13, GRK-GFP, GRK- RlucFI , RlucF2-Gy5, GRK2-GFP-mem, Rluc-GRK2, GFP-G_Y5, GFP-CAAX or GPCR-Rluc.

MODE(S) FOR CARRYING OUT THE INVENTION

The present invention is illustrated in further details by the following non-limiting examples.

Example 1 : Materials and Methods

Reagents. Angiotensin II (Angll; [Asp-Arg-Val-Tyr-lle-His-Pro-Phe], SEQ ID NO: 49), poly-ornithine, poly-D-lysine, isoproterenol, rotigotine, epinephrine, norepinephrine, phenylephrine and Pertussis toxin were from Sigma®. u46619 were from Cayman Chemical® (Ann Arbor, Ml). [Sar , lle⁸]-Angll (SI) and [Asp ,Val⁵, Gly⁸]-Angll (DVG) [Sar1 -Val5-D-Phe8] Angll (SVdF) and [Sar1-D-Ala8] Angll were synthesized at the Universite de Sherbrooke (Canada, QC). UBO-Qic (L-threonine,(3R)-N-acetyl-3-hydroxy-L-leucyl-(aR)-a- hydroxybenzenepropanoyl-2,3-idehydro-N-methylalanyl-L-alanyl-N-methyl-L-alanyl-(3R)-3- [[(2S,3R)-3-hydroxy-4-methyl-1-oxo-2-[(1 -oxopropyl)amino]pentyl]oxy]-L-leucyl-N,0-dimethyl- ,(7→1)-lactone (9CI)) was obtained from Institute for Pharmaceutical Biology of the University of Bonn (Germany). Dulbecco's modified Eagles medium (DMEM), fetal bovine serum, OPTI- MEM®, and other cell culture reagents were purchased from Invitrogen®. Coelenterazine 400a, Coelenterazine H and Prolume® Purple I were purchased from either Goldbio®, Biotium® or Nanolight® Technology. Polyethylenimine (PEI ; 25 kDa linear; was purchased from Polysciences® (Warrington, PA, USA). Salmon sperm DNA was purchased from Lifetechnologies (ThermoFisher). Phusion DNA polymerase was from Thermo Scientific®. Restriction enzymes and T4 DNA ligase were obtained from NEB®. Oligonucleotides for mutagenesis and PCR applications were synthetized at BioCorp DNA®.

Expression vectors: Receptors and G-proteins. The plasmid encoding AT1 R was a generous gift from Stephane Laporte ( cGill University, Montreal, Canada). Ga_q, Ga-n , Ga₁₂, Ga₁₃, Ga₁₄, Ga_15/16, Ga_oA, Ga_oB, Ga_z, Ga_s, Ga_n , Ga_i2, Ga_i3, Gp1 , TPaR, D₂R and a_1BAR were obtained from the cDNA Resource Center (cDNA.org). Plasmids encoding mutant Ga proteins including Ga_qG66K, Ga_qY67C and Ga_qF75G, were obtained by site-directed mutagenesis (PCR overlap) of the Ga_q wild-type protein coding sequence using the primers depicted in Table I . The PCR fragments were digested with Acc65\ + Xho\ restriction enzymes and cloned in pCDNA3.1 Zeo(+) (from Invitrogen®, Carlsbad, California) digested Acc65\ + Xho\. DNA sequencing was used for validation of the different constructs and to identify the specific substitutions created from degenerated primers.

Table I: Sequences of primers used in the experiments described herein

Expression vectors: Biosensor constructs. Rluc-Gy5 and GFP-Gy. Plasmid encoding the fusion proteins Rluc-Gy1 to Gy13 and GFP1 0-Gy5 were was obtained by PCR amplification of the Gy coding sequences which were then fused in frame at its N-terminus to the humanized Renilla luciferase II (hRlucl l) sequence (a variant of the hRluc previously reported (Leduc, Breton et al. 2009), SEQ ID NO:39) into pcDNA3.1 vector (linker sequence: GSAGT, SEQ ID NO: 33) , or to the GFP10 (a variant form of the green fluorescent protein (GFP) previously reported (Mercier, Salahpour et al. 2002, SEQ ID NO:38). GRK2-GFP and GRK3-GFP: GRK2- GFP, GRK3-GFP, GRK2 Cterm (SEQ ID NO:50)-GFP, GRK3 Cterm (SEQ ID NO:51 )-GFP were generated by PCR amplification of GRK2 and GRK3, which were then fused at their C- terminus to the GFP10 into pcDNA3.1 Zeo(+) vector, generating a linker of 1 1 amino acid residues between the GRK and the GFP10 protein (linker sequence: GSAGTGKLPAT, SEQ ID NO: 34). GFP-GRK2 and GFP-GR 3: GRK2-GFP, GFP-GRK2 Cterm (SEQ ID NO:50), GFP- GRK3 Cterm (SEQ ID NO:51 ) were generated by PCR amplification of GRK2 and GRK3, which were then fused at their N-terminus to the GFP10 (SEQ ID NO:38) into pcDNA3.1 Zeo (+) vector, generating a linker of 7 amino acid residues between the GRK and the GFP10 protein (linker sequence: GSAGTGG, SEQ ID NO:52). GFP- and Rlucl l-tagged GRK2 mutants were generated by PCR-directed mutagenesis using a similar procedure. GRK2-Rluc F1 and Rluc F2-Gy5: The GRK2-Rluc F1 was obtained by PCR amplification of the coding sequence for residues 1 to 1 10 from the humanized Renilla luciferase I I sequence set forth in SEQ ID NO:39 (Rluc F1), which was subsequently fused to the C-terminus of the GRK2 protein in the pcDNA3.1 Zeo (+) vector, generating a 18 amino acids linker between the Rluc fragment and the GRK2 (linker sequence: GSAGWGKLGSAGSGSAGS, SEQ ID NO:35). The Rluc F2-Gy5 was obtained by PCR amplification of the coding sequence for residues 1 1 1 to 31 1 from the humanized Renilla luciferase sequence set forth in SEQ ID NO:39 (Rluc F2), which was subsequently fused in frame of the N-terminus of the Gy5 protein into the pcDNA3.1 Zeo(+) vector, generating a 1 1 amino acid residues linker between the Rluc fragment and the Gy5 (linker sequence: GSAGTGSAGTT, SEQ ID NO:36). GRK2-GFP-mem: The GRK2-GFP-mem construct encoding a fusion protein between the GRK2-GFP and a 200 amino acid residues flexible linker followed by the membrane anchoring signal of the human KRAS protein (prenylation motif: CAAX) (Hancock 2003) was generated as follows. First, a linker with a predicted disordered structure was created from a random sequence of 2000 residues. From this sequence, a segment of 200 residues with minimal globularity and maximum disorder index was selected, after elimination of aggregation hotspots, putative localization, interaction and phosphorylation motifs. This 200-amino acid flexible linker (SEQ ID NO:53) was directly synthesized and then fused in frame at the N-terminus of the membrane anchoring signal of human KRAS protein splice variant b (amino acid sequence: KKKKKKSKTKCVIM, SEQ ID NO:37) using PCR amplification. The flexible linker followed by KRAS prenylation signal was then sub-cloned into the GRK2-GFP pcDNA3.1 Zeo(+) vector, at the C-terminus of the GRK2- GFP protein . Polycistronic biosensor vector: The polycistronic vector encoding GRK2-GFP, Rluc-Gy5 and ΰβ1 was developed by first sub-cloning the WT and D1 10A mutant GRK2-GFP10 fusion proteins into the pLVX vector. Then, sub-cloning of IRES-Gp1 into pcDNA3.1 Rluc-Gy5 was performed to obtain pcDNA3.1 Rluc-Gy5-IRES-Gpi . Finally, the two constructs were assembled to generate a pLVX vector containing GRK2-GFP-IRES-Rluc-Gy5-IRES-Gpi . rGFP- CAAX: Plasmid encoding the fusion protein rGFP-CAAX was obtained by PCR amplification of rGFP coding sequence (SEQ ID NO:46) with a reverse primer encoding a linker (sequence: GSAGTMASNNTASG , SEQ ID NO:47) and the plasma-membrane targeting polybasic sequence and prenylation signal sequence from KRAS splice variant b: -GKKKKKKSKTKCVI (named: CAAX, SEQ ID NO:37) . The CAAX plasma-membrane targeting sequence is in frame at the C-terminus of the rGFP coding sequence. The PCR fragment is sub-cloned into pcDNA3.1 (+) vector. Rlucll-GRK2: The GRK2 cDNA was PCR-amplified and subcloned with Rlucl l at its N-terminus in plREShyg3 expression vector (from Clonetech®) with the linker: GGSGSGSGS (SEQ ID NO:48).

Cell culture and transfections. Human embryonic kidney 293 (HEK293) cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum, 100 unit/ml penicillin/streptomycin at 37°C in a humidified atmosphere with 5% C0₂. Two days before the experiments, HEK293 cells were transfected with the indicated plasmids using poly-ethylenimine 25-kDa linear (PEI) as a transfecting agent (at a ratio of 3 to 1 , PEI/DNA) (Hamdan, Rochdi et al. 2007) , and then directly seeded in 96-well plates pre-treated with poly-L-ornithine hydrobromide or Poly-D-Lysine, at a density of 35,000 cells per well (for BRET and PCA assays in living cells), or 6-well plates at a density of 1 ,000,000 cells per well (for BRET assays on membrane preparations) .

BRET assays in living cells. Cells seeded in 96-well plates were washed twice with

Phosphate Buffered Saline (PBS), followed by Tyrode buffer addition (composition: 137 mM NaCI, 0.9 mM KCI, 1 mM MgCI₂, 1 1 .9 mM NaHC0₃, 3.6 mM NaH₂P0₄, 25 mM HEPES, 5.5 mM Glucose and 1 mM CaCI₂, pH 7.4). The cells were then treated with the different ligands or vehicle for the indicated times. The luc substrate, coelenterazine 400a, was added at a final concentration of 2.5 μΜ and cells were further incubated for an additional 5 minutes. BRET values were then collected using a Mithras™ LB940 Multimode Microplate Reader or a TRISTAR® LB942 Multimode Microplate Reader, equipped with the following filters: 400 nm ± 70 nm (energy donor) and 515 nm ± 20 nm (energy acceptor). BRET values were determined by calculating the ratio of the light emitted by GFP (515 nm) over the light emitted by the Rluc (400 nm). To determine the % of activation (Stim as % of basal), BRET values obtained for the agonist treated cells where expressed as a percentage of the BRET values obtained with the corresponding cells treated with vehicle.

BRET assays for GRK2-GFP translocation to Rlucll-tagged receptor (FIGs. 15B and 15C): 100 ng of HA-TPaR-Rlucll , 750 ng of GRK2-GFP10 (WT in FIG. 15B or D1 10A mutant in FIG. 15C), 100 ng of the indicated Ga, 100 ng of WT Gp1 and 100 ng of WT Gy5 and PEI at a ratio of PEI :DNA of 3: 1 , is added to a suspension of HEK293SL (350,000 cells/ml). Cells were seeded (100 μΙ of cells/PEI/DNA suspension per well of a 96-well plate) on poly-D-lysine pretreated plates. 48 h post-transfection, cells were washed and preincubated in Tyrode + 1 m CaCI₂ at 37°C for 60 min. Cells were exposed for a total of 15 min to different doses of U-46619 in FIG. 15B and 15C, at 37°C. Coelenterazine 400a was then added at a final concentration of 2.5 μ within the last 5 min of stimulation. BRET was measured at 37°C, using a Tristar® Microplate Reader (Berthold Technologies®) .

BRET assays for Rlucll-GRK2 translocation to the plasma-membrane labeled with rGFP-CAAX (Kras) (FIGs. 16B and 16C): 100 ng of HA-TPaR, 20 ng of Rlucll-GRK2, 100 ng of the indicated Ga, 100 ng of WT Gpi , 100 ng of WT Gy5, 400 ng of rGFP-CAAX (Kras), 180ng of ssDNA, and PEI at a ratio of PEI:DNA of 3: 1 , is added to a suspension of HEK293SL (350,000 cells/ml). Cells were seeded (100 μΙ of cells/PEI/DNA suspension per well of a 96-well plate) on poly-D-lysine pretreated plates. 48 h post-transfection, cells were washed and preincubated in Tyrode + 1 mM CaCI₂ at 37°C for 60 min. Cells were exposed for a total of 15 min to different doses of U-46619 in FIG. 16B or for Z' Factor determination (FIG. 16C) to either vehicle or 100 nM U46619 was added to the wells of half of a 96-well plate, at 37°C. Coelenterazine 400a was then added at a final concentration of 2.5 μΜ within the last 5 min of stimulation. BRET was measured at 37°C, using a Tristar® Microplate Reader (Berthold Technologies). Z' Factor determination was obtained as described previously.

G-protein inhibitors. BRET assays were performed as described previously, except that cells were pre-treated overnight at 37°C with 100 ng/ml of pertussis toxin, or, for 20 minutes at 37°C with 100 nM of Ubo-Qic.

Kinetics experiments. BRET assays were performed as described previously, except that BRET readings were collected at regular intervals, 5 min after coelenterazine addition, while ligands and vehicle were injected to the cells after 30 sec of BRET measurements.

Z'-factor determination. HEK293 cells were transfected as described with the indicated constructs (see description of FIGs 7A, 7B, 9A, 10C, 11 D and 16C). BRET assays were performed as described previously, with half of the 96-well plate treated with the indicated agonists and the second half of the plate treated with the corresponding vehicle. Z'-factor were calculated as described by Zhang et al. (Zhang, Chung et al. 1999). A Z'-factor between 0.4 and 1 is considered a robust assay.

Protein complementation assays using Rlucll fragments. Cells were washed twice with

PBS, followed by Tyrode buffer addition. The cells were then pre-treated with the Rluc substrate, coelenterazine 400a, at a final concentration of 2.5 μΜ for 30 min at 37°C. The different ligands or vehicle were added for an additional 10 min. Luminescence values were then collected using a Mithras™ LB940 Multimode Microplate Reader, without any filters.

BRET assays on membrane preparations. Cells seeded in 6-well plates were collected, re-suspended in lysis buffer (composition: 25 mM Tris-HCI pH 7.4, 2 mM EDTA, 5mM MgCI₂, 27% sucrose, 15 μΜ GDP, 2 μΜ GTP, 10 μg/ml benzamidine, 5 μg/ml soybean trypsin inhibitor and 5 g/ml leupeptin) and subjected to a polytron homogenization. Following centrifugation steps, the membrane pellets were resuspended in Tyrode buffer supplemented with 5 mM MgC , 15 μΜ GDP and 15 μΜ GTP. BRET experiments were then performed as described previously, using 400 pg of membrane per well.

BRET Titrations (in FIGs. 2B and 2C): HEK293 cells were transiently transfected using

PEI in a pg DNA to μΙ PEI 1 mg/ml ratio equal to 1 g:3 l. The DNA transfected per well of a 96- well plate is as follows: In FIGs. 2A and 2B: 40ng of HA-TPaR or HA- iAR, 0.5 ng of Rlucll- Gy5, 10 ng Ga-n or Ga₁₅, 10 ng Gp1 encoding constructs and an increasing quantity of GRK2 constructs tagged with GFP10, up to 75 ng. The BRET assay was performed 2 days post- transfection; cells were washed once with PBS and left in Tyrode's buffer. The cells were treated with vehicle or agonist drug, 100n U-46619 (FIG. 2B) or 1 μ isoproterenol (FIG. 2A) for a total of 15 min at RT. The Rluc substrate Coel-400a was then added at a final concentration of 2.5 μΜ within the last 5 min of stimulation. BRET values were then collected using a Mithras® LB940 Multimode Microplate Reader, and determined by calculating the ratio of the light emitted by the acceptor over the light emitted by the Rlucll . The titration curves (FIGs. 2B and 2C) represent the BRET ratios obtained in function of GFP-construct expression (evaluated in fluorescence) over Rlucl l construct expression (evaluated in bioluminescence).

Example 2: Results

To study the activation of specific G-proteins by GPCRs, an assay was developed based on the competition between Ga subunits and βγΙΡ for their binding to Θβγ subunits. As depicted in FIGs. 1A and 1 B, in the absence of receptor activation, the Ga subunit is tightly bound to the ΰβγ dimer, preventing its association with the βγΙΡ. Following receptor stimulation, the GTP-bound Ga dissociates from the G y complex, which is then free to interact with the βγΙΡ. The interaction between the βγΙΡ and the ββγ, therefore, reflects the G-protein activation. By co-expressing βγΙΡ and ΰβγ, each tagged with one of the two components of the detection system, with different subtypes of untagged Ga, it has been possible to determine the coupling profile of a given receptor following its activation. As shows below, different methods of detection may be used to assess the interaction between the βγΙΡ and the Θβγ, such as resonance energy transfer (RET) approaches (FIG. 1A: bioluminescence (BRET) or fluorescence resonance energy transfer (FRET)) ; or FIG. 1 B: protein complementation (PC) assays.

Taking RET as an example of detection method, possible scenarios and corresponding results interpretation for βγΙΡ-based biosensors of G-protein activation are shown in FIG. 1 C. In the absence of any Ga subunit co-transfected with the two RET partners βγΙΡ-Α and ΰβγ-ϋ, the basal RET signal is relatively high due to the constitutive interaction between βγΙΡ and ΰβγ dimer. In that case, a modulation of the RET signal measured following receptor stimulation would reflect the activation of endogenous Ga subunits (Mock or -Ga condition). Co-expression of a Ga subunit prevents the basal interaction between βγΙΡ-Α and Gpy-D, leading to a decrease in the basal RET response recorded (white bars in +G0₁ and +G<¾ conditions). Upon stimulation of the receptor (with a suitable GPCR ligand, for example), significant increase in the modulation of the RET signal as compared to the mock condition is observed only if the receptor engages (i.e. is coupled to) the specific Ga subunit co-expressed with the other biosensor components (black bar, +Ga₂ conditions). However, if the over-expressed Ga subunit is not functionally coupled to the receptor, no significant change in the BRET signal is detected upon receptor stimulation (black bar, +Gai conditions).

FIGs. 2A to 2C present some of the different constructs tested for optimization of the βγΙΡ-based G-protein activation biosensor. Four different GFP-tagged constructs for GRK2 and 3 were tested, two based on the complete GRK coding sequence and two on the C-terminal PH domain/Gp binding domain, with GFP at either the N-terminal or the C-terminal portion of GRK (FIG. 2A). The results presented in FIGs. 2B and 2C indicate that all GRK2 configurations/constructs gave a detectable BRET response (and thus may be used in the biosensor), and that the full-length GRK2 tagged at its C-terminal with a BRET acceptor (e.g., GFP) is giving the best dynamic window in term of amplitude of BRET signal and stability of response over a wider range of donor to acceptor ratios. Similar results were obtained using GFP-tagged GRK3 constructs.

To assess the feasibility of using a βγΙΡ to monitor G-protein activation, the GRK2 protein, which specifically interacts with free Gpy dimers, was selected as a representative βγΙΡ, and tagged at its C-terminus with the energy acceptor GFP10 (GFP), thus allowing the use of BRET as a readout of its interaction with G y. The GRK2-GFP fusion protein was co-expressed with a G 5 subunit tagged in N-terminus with the energy donor Renilla luciferase (Rluc), as well as with untagged G 1 and Ga subunits. In addition to the biosensor components (GRK2-GFP and Rluc-Gy5), G 1 and various Ga, cells were co-transfected with the thromboxane A2 receptor (TPaR), which was chosen as an example of a prototypical GPCR. In the experiment depicted in FIG. 3A, the G-protein coupling profile of the TPaR was determined by stimulating cells co-expressing the different Ga proteins, Ga_q, Ga_{11 :} Ga ₂, Ga ₃, Ga ₄, Ga ₅, Ga_oA, Ga_oB, Ga_z, Ga_s, Ga_n , Ga_i2 and Ga_i3, with the TPaR agonist U-46619 (a stable synthetic analog of the endoperoxide prostaglandin PGH2), and compared with results obtained in absence of Ga over- expression (mock condition, left bars). In the absence of Ga co-transfection, the BRET signal recorded was relatively high and only slightly modulated upon stimulation with U-46619, reflecting activation of endogenous Ga proteins. Upon co-expression of specific Ga proteins, an agonist-induced modulation of the BRET signal was significantly higher in cells over-expressing Ga_q, Ga-ii , Ga ₂, Ga ₃, Ga-|₄ and Ga_15/16, relative to the mock condition or to cells over- expressing Ga subunits of the Ga, family, indicating that TPaR is coupled to the activation of G- proteins of the Ga_q and Ga-|₂ families, but not of those of the Ga, family (FIG. 3B).

In addition to the wild-type (native) Ga proteins, three Ga_q mutants (Ga_qG66K, Ga_qY67C and Ga_qF75G , were also used in the panel of G-proteins tested with the TPaR. The substitution of the glycine residue at position 66 of the Ga_q protein for a charged residue (GqG66K for example), had been previously described as resulting in Ga_q protein mutants with promiscuous coupling properties, as they can also be activated by non-Ga_q-coupled receptors (Heydorn, Ward et al. 2004). As can be seen on FIGs. 3A and 3B, the previously described Ga_qG66K mutant, as well as the novel Ga_qY67C and Ga_qF75G described herein, were activated by the TPaR. These promiscuous Ga proteins (or any Ga proteins having equivalent mutations at these positions, see FIG. 14) may be used as positive controls for GPCR activation in βγ- based biosensor assays and be particularly useful with receptors for which only limited information is available on their coupling preferences, such as orphan receptors. Ga_q, Ga^, Ga-i4, Ga _{5/ 6}, Ga_qG66K and Ga_qY67C were then selected for dose-response curves of U-46619 (FIG. 3C). Interestingly, potencies ranging from 0.5 nM for G 13 to 6.6 nM for Ga_15/16 were measured, validating that βγΙΡ-based biosensor assays can detect the specific potency of activation linked to each G-protein engaged by a given pair of receptor-ligand.

To further illustrate that a βγΙΡ-based G-protein activation biosensor can be used to reveal the specificity of G-protein activation for different GPCRs, the dopamine D2 receptor (D₂R) and the a_{1 B}-adrenergic receptor (a_{1 B}AR) were each co-expressed with GRK2-GFP, Rluc- Gy5, Gp1 and various Ga, and stimulated with their prototypical agonists; rotigotine for D₂R, and phenylepinephrine for a_1BAR. As shown in FIGs. 4A and 4C, each receptor displays a specific G-protein activation profile, distinct from the one observed with TPaR (FIGs. 3A and 3B); D₂R is solely coupled to Ga_rfamily members (Ga_oA, Ga_oB, Ga_z, Ga^ , Ga_i2 and Ga_i3), while a _BAR is exclusively coupled to Ga_q-family members (Ga_q, Ga-n and Ga_15/16). The promiscuous Ga_q mutant Ga_qY67C was activated by the two receptors (FIGs. 4A and 4C) . Dose-response curves were obtained with some of the G-proteins activated by D₂R (Ga_n and Ga_qY67C) and a _BAR (Ga_q and Ga-n) (FIGs. 4B and 4D). In FIG 4E, dose-response curves of Ga_z activation were obtained for different adrenergic agonists: epinephine, norepinephrine, phenylephrine and isoproterenol, from HEK293 cells expressing a_2CAR, Ga_z, GRK2-GFP, Rluc-Gy5 and GB1 . These results demonstrate that a βγΙΡ-based G-protein activation biosensor may be used to establish G-protein preferences and activation/pharmalogical profiles of GPCRs, as well as a pharmacological tool to address potencies and efficacies of given ligands for activating various G-proteins through their cognate receptors. As shown in FIGs. 4F to 4J, distinct G-protein activation profiles were obtained with different combinations of G and Gy subunits. The results show that combinations of both Gp and Gy subunits can lead to distinct pharmalogical profiles of G-protein activation. These differences could, at least in part, be linked to distinct pharmalogical profiles observed with different cells and tissues expressing not only a specific set of Ga subunits but also different combination and levels of G(3 and Gj subunits.

Inhibitors of G-protein activity such as pertussis toxin (PTX) and Ubo-Qic (structurally related to the cyclic depsipeptide Y -254890) which selectively blocks Ga, and Ga_q activation, respectively, have been extensively used in the field of GPCRs to characterize the coupling properties of receptors (Takasaki, Saito et al. 2004) . Using the βγΙΡ-based G-protein activation biosensor, experiments were performed with those selective G-protein inhibitors to demonstrate the specificity of the BRET signals obtained. For the TPaR, which is coupled to Ga_q activation, the BRET response measured using βγΙΡ-based biosensor following agonist stimulation, was completely abolished upon Ubo-Qic pre-treatment, while PTX pre-treatment had no effect on this response (FIG. 5A). In contrast, for the Ga_rcoupled receptor D₂R, PTX pre-treatment significantly reduced the biosensor BRET response detected after agonist incubation, whereas pre-treatment with the Gq inhibitor Ubo-Qic had no detectable effect on the BRET signal recorded (FIG. 5B) . TPaR-mediated G-protein activation was also used to validate Ubo-Qic inhibitor selectivity. The results presented in FIG. 5C show that, from the Ga_q familly (Ga_q, Ga-n , Ga-i4 and Ga _{5/ 6}) , only Ga _{5/ 6} is insensitive to Ubo-Qic. The Ga₁₂ and Ga ₃ proteins are also insensitive to Ubo-Qic. Finally, the βγΙΡ-based biosensor was used to reveal the Ubo-Qic sensitivity of the activation of mutant Ga_q (at position 67 or 75 - see FIG. 14). Substitutions of the tyrosine residue at position 67 (Y67C, Y67G, Y67S & Y67L) led to resistance to Ubo-Qic and promiscuous properties, indicating that this residue may be important for controlling G- protein activation, and that the substitution of residue Phe75 to glycine (which is associated with a promiscuous phenotype - see FIG. 4A) led to a partial Ubo-Qic mediated inhibition of activation (FIG. 5D) Thus, in addition to validating the specificity of the BRET signals recorded for the various G-proteins, these results support the use of βγΙΡ-based G-protein activation biosensors described herein as tools to identify/develop novel selective inhibitors of G-proteins.

To further characterize the βγΙΡ-based G-protein activation biosensor, kinetics of Ga_M (FIG. 6A) and Ga-n (FIG. 6B) activation were determined following agonist treatment of D₂R and TPaR, respectively. As shown in FIGs. 6A and 6B, similar kinetics of activation were obtained for the two different receptors and G-proteins, with a maximal response reached approximately 30 seconds after ligand addition and a plateau lasting at least 30 minutes following the initial stimulation. This sustained response is particularly well suited for assay adaptation to high- throughput screening (HTS).

To evaluate the robustness of the assay, Z'-factors were determined for G-protein activation through typical Ga,- (D₂R, FIG. 7A) and Ga_q- (TPaR, FIG. 7B) coupled receptors. The assay is particularly robust with Z'-factors of 0.79 and 0.89 for D₂R/Ga_M (FIG. 7A) and TPaR/Ga-ι-ι (FIG. 7B), respectively. The robustness of this assay is compatible with the requirements of screening applications, notably HTS applications. In addition to the previously described potential applications of the βγ-based biosensor in G-protein profiling of receptors and HTS, ligand characterization represents another application of this G-protein activation biosensor. GPCRs can preferentially engage different G- proteins and signaling pathways upon activation with different ligands, this phenomenon is known as ligand-biased signaling of GPCRs (Galandrin, Oligny-Longpre et al. 2007) (Kenakin and Christopoulos 2013) . The biosensors described herein are particularly well suited for performing ligand profiling experiments since it is possible to assess the activity of all G-protein subtypes using the same RET partners. As a representative example, various ligands of the angiotensin II type 1 receptor (AT1 R) were profiled using the βγΙΡ-based G-protein activation biosensor (FIGs. 8A to 8C). A first set of experiments was performed to determine the coupling properties of the receptor using its natural ligand, angiotensin I I. As shown in FIG. 8A, the AT1 R is coupled to several members of the Ga_q-, Ga₁₂- and Ga_rfamily of proteins. Ga_q, Gan and Ga₁₂ were then selected for further characterization following activation of AT1 R with different analogs of angiotensin I I . As shown in FIGs. 8B and 8C, those angiotensin II derived-peptides stimulated the different G-proteins to various extents, revealing a potential bias of some ligands toward specific G-proteins. For example, the DVG peptide showed a better efficacy for Gcd₂ activation than Ga_q, relative to angiotensin II response (FIG. 8C).

It was next assessed whether protein complementation assay (PCA) , instead of RET- based assays, may be used to assess the interaction between the βγΙΡ and the Gpy subunits (FIG. 1 B). PCA, such as bimolecular fluorescence complementation (BiFC) or enzyme fragment complementation (EFC) , allows the detection of interaction between two protein partners, which makes it compatible with the βγΙΡ-based G-protein activation biosensor. To determine whether it is possible to use an EFC-based assay, and more particularly an Rluc-based EFC assay (Stefan, Aquin et al. 2007), in the βγΙΡ-based G-protein activation biosensor, two fusion proteins were generated: GRK2 tagged with an N-terminal portion of Rluc in C-terminus (GRK2-Rluc F1 ) , and the complementary C-terminal portion of Rluc in N-terminus of Gy5 (Rluc F2-Gy5). If an interaction between GRK2 and the free ΰβγ subunits occurs (following G-protein activation), the two complementary Rluc fragments would re-associate and luminescence can be measured in presence of the Luc substrate coelenterazine. A proof of concept was done using cells co- expressing TPaR with GRK2-Rluc F1 , Rluc F2-Gy5, ΰβ1 and Ga^ . A robust luminescent signal was measured following stimulation with U-46619, revealing G1 1 protein activation, with a Z'- factor of 0.53 (FIG. 9A) and an EC₅o of 8.4 n (FIG. 9B), validating that PCA may be used in the βγΙΡ-based G-protein activation biosensor described herein.

It was next assess whether βγΙΡε other than GRK2 (such as GRK3) could be used to monitor G-protein activation in the βγΙΡ-based G-protein activation biosensor. A fusion protein was generated between GRK3 and the energy acceptor GFP, and the resulting GRK3-GFP was co-expressed with Rluc-Gy5, Gp1 , Ga_n and the D₂R, to obtain dose-response curves of dopamine. As seen in FIG. 10A, similar potencies were observed using GRK2 or GRK3-based biosensors (96 pM for GRK2 and 56 pM for GRK3). The kinetics of activation of the GRK3- based biosensor were also similar to those obtained using the GRK2-based biosensor (FIGs. 6A and 6B), with a maximal response reached at approximately 30 seconds, and a plateau of at least several minutes (FIG. 10B). Finally, Z'-factor were also generated with the GRK3-based biosensor. Using the D₂R and Ga_n , a Z'-factor of 0.71 was obtained with the GRK3-biosensor, confirming the robustness of the assay (FIG. 10C). Taken together, these data demonstrate that different βγΙΡ can be used in the βγΙΡ-based G-protein activation biosensor to assess G-protein activation.

To simplify the use of the βγΙΡ-based G-protein activation biosensor, a polycistronic vector encoding the GRK2-GFP, Rluc-Gy5 and Gp1 was developed (FIG. 11 A). This ensures that the components of the biosensor are expressed from a single construct and at a fixed ratio, which could minimize inter-experiment variability. As shown in FIG. 11 D, a Z'-factor of 0.8 was obtained using the polycistronic construct, co-transfected with plasmids encoding for TPaR and Gan. This result is comparable to the Z'-factor obtained with cells transfected with plasmids encoding individual biosensor components (FIG. 7B: Z' = 0.89). Dose-response curve experiments were also performed using this polycistronic vector and an EC₅₀ value of 4.3 n was obtained for the TPaR stimulated with U-46619 (FIG. 11 C), similar to the EC₅₀ of 8.4 nM measured for the same receptor/ligand pair used with the Rluc-PCA based GRK2 biosensor (FIG. 9B). These results confirm the validity of expressing the different components of the biosensor in a polycistronic vector, which could be advantageously used to establish stable cell lines with only one selection marker, thus simplifying the experimental procedures and potentially improving reproducibility (e.g., minimizing inter-experiment variability).

Another variant of the βγΙΡ-based G-protein activation biosensor was developed, in which the GRK2 protein is tethered at the plasma membrane (PM) (FIG. 12A). This construct may be useful for some specific applications where in vitro experiments on membrane preparations would be preferred to whole cell experiments, such as for screening applications. To validate this approach, BRET experiments where performed on membrane preparations expressing TPaR, Gan , Οβ1 , Rluc-Gy5, and either the cytoplasmic form of GRK2-GFP used previously (GRK2 wt in FIG. 12B) or the plasma membrane anchored GRK2-GFP (GRK2- mem). A superior modulation of the BRET signal was observed with the membrane tethered GRK2, relative to the wt GRK2 (FIG. 12B), for which only marginal BRET increase was detected upon ligand stimulation. These results validate the use of a PM-anchored βγΙΡ to measure G- protein activation on membrane preparations.

The results depicted in FIGs. 13A to 13C show that mutations reported to affect GR 2 functions, such as the D1 10A substitution in the RGS domain (RGS-dead mutant) and the K220R substitution in the catalytic domain (catalytic-dead mutant) , or its regulation by phosphorylation (such as the S670A, S676A and S685A substitutions, or the S670D, S676D and S685D substitutions, that respectively prevent and mimic phosphorylation of GRK2's C- terminal binding domain by ERK, PKA and CDK2-CyclinA, do not prevent nor significantly promote its recruitment to activated G-proteins, as assessed using a βγΙΡ-based G-protein activation biosensor. GRK2 variants comprising the above-noted mutations are recruited to a similar extent as native GRK2 (FIGs. 13A to 13C), thus providing evidence that GRK2 recruitment to ΰβγ could be insensitive to regulation by different signalling events. Similar results were obtained with the GRK2 D1 10A mutant following activation of AT1 R with angiotensin I I .

Another biosensor to measure the competition between Ga subunits and βγΙΡ for their binding to σβγ subunits was developed; FIG. 15A shows the configuration and principle of such biosensor. The biosensor comprises a βγΙΡ (GRK) tagged with a RET donor or acceptor (a RET acceptor (A) is illustrated) and a GPCR tagged at its C-terminal with a RET donor or acceptor (a RET donor (D) is illustrated). While in the inactive form, the Ga subunit of the heterotrimeric G- protein is tightly bound to the G y dimer. Upon ligand (L) binding to the GPCR, the Ga dissociates from the G y subunits, allowing βγΙΡ to be recruited to the free σβγ subunits and bringing the BRET acceptor in close proximity to the BRET donor RLuc linked to the GPCR, thus inducing/increasing the BRET signal. FIGs. 15B and 15C show dose-response curves for G-protein activation, obtained with a biosensor according to FIG. 15A, comprising a wild-type GRK2 (FIGs. 15A) or the RGS-dead GRK2 mutant (D1 10A) (FIG. 15B). The dose-response curves showed similar profiles in FIGs. 15B and 15C indicating that a functional RGS is not required to recruit a βγΙΡ to an activated G-protein, confirming the results presented in FIGs. 11 B and 13A using a different biosensor configuration.

Another biosensor to measure the competition between Ga subunits and βγΙΡ for their binding to ββγ subunits was developed; FIG. 16A shows the configuration and principle of such biosensor. The biosensor comprises a βγΙΡ (e.g., GRK) tagged with a RET donor or acceptor (a RET donor (D) is illustrated) and a plasma membrane (PM)-targeting domain tagged with a RET donor or acceptor (a RET acceptor (A) is illustrated). While in the inactive form, the Ga subunit of the heterotrimeric G-protein is tightly bound to the G y dimer. Upon ligand (L) binding to the GPCR, the Ga dissociates from the σβγ subunits, allowing βγΙΡ to be recruited to the free ΰβγ subunits that are located at the PM, and bringing the RET donor D in close proximity to the RET acceptor A anchored to the PM, thus inducing/increasing the BRET signal. FIG. 16B shows dose-response curves for G-protein activation, obtained with a biosensor according to FIG. 16A, using HEK293 cells co-expressing TPaR, different Ga (Ga_q = solid square, Ga^ = solid triangle, Mock condition (no Ga) = empty circle), ΰβ1 , Gy5, Rlucl l-GRK2 and rGFP-CAAX, stimulated with increasing doses of U46619. The dose-response curves in FIG. 16B are similar to those obtained with biosensors having a different configuration (FIGs. 3C, 9B and 11 C), providing evidence that a biosensor measuring βγΙΡ recruitment at the PM is suitable to "indirectly" assess βγΙΡ recruitment to the free Θβγ subunits that are anchored to the PM.

Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims. In the claims, the word "comprising" is used as an open-ended term, substantially equivalent to the phrase "including, but not limited to". The singular forms "a", "an" and "the" include corresponding plural references unless the context clearly dictates otherwise.

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Claims

WHAT IS CLAIMED IS:

(A)

(i) a first biosensor comprising:

a second component comprising a fused Gp protein or a fused Gy protein, wherein said Gp protein or said Gy protein is fused to (a) a RET donor; (b) a RET acceptor or

(c) a second fragment of said reporter protein;

(ii) a second biosensor comprising:

the first and second components defined in (i); and

a third component comprising a recombinant Ga protein;

wherein (a) if said βγΙΡ is fused to said RET donor, said Gp or Gy protein is fused to said RET acceptor; (b) if said βγΙΡ is fused to said RET acceptor, said GB or Gy protein is fused to said RET donor; and (c) if said βγΙΡ is fused to said first fragment of said reporter protein, said Gp or Gy protein is fused to said second fragment of said reporter protein; or

(B)

(i) a biosensor comprising

a third component comprising a recombinant Ga protein;

wherein (a) if said βγΙΡ is fused to said RET donor, said GPCR is fused to said RET acceptor; (b) if said βγΙΡ is fused to said RET acceptor, said GPCR is fused to said RET donor; and (c) if said βγΙΡ is fused to said first fragment of said reporter protein, said GPCR is fused to said second fragment of said reporter protein.

2. The biosensor system of claim 1 , wherein said Gy protein is fused to said RET donor, RET acceptor or second fragment.

3. The biosensor system of claim 1 or 2, wherein said RET donor, RET acceptor or second fragment is fused at the N-terminus of said ΰβ or Gy protein.

4. The biosensor system of any one of claims 1 to 3, wherein said RET donor, RET acceptor or first fragment is fused at the C-terminus of said βγΙΡ.

5. The biosensor system of any one of claims 1 to 4, wherein said βγΙΡ is fused to said RET acceptor and said G protein, Gy protein or GPCR is fused to said RET donor.

6. The biosensor system of any one of claims 1 to 5, wherein said RET donor is a bioluminescent protein.

7. The biosensor system of claim 6, wherein said bioluminescent protein is a luciferase.

8. The biosensor system of claim 7, wherein said luciferase is a Renilla luciferase.

9. The biosensor system of any one of claims 1 to 8, wherein said RET acceptor is a fluorescent protein.

10. The biosensor system of claim 9, wherein said fluorescent protein is a GFP.

1 1 . The biosensor system of any one of claims 1 to 4, wherein said βγΙΡ is fused to said first fragment, and said Θβ protein, Gy protein or GPCR is fused to said second fragment.

12. The biosensor system of claim 1 1 , wherein said reporter protein is a bioluminescent protein.

13. The biosensor system of claim 12, wherein said bioluminescent protein is a luciferase.

14. The biosensor system of claim 13, wherein said luciferase is a Renilla luciferase.

15. The biosensor system of claim 14, wherein said first fragment comprises about residues 1 to 1 10 of Renilla luciferase, and said second fragment comprises about residues 1 1 1 to 31 1 of Renilla luciferase.

16. The biosensor system of any one of claims 1 to 15, wherein the first component further comprises a plasma membrane (P )-targeting moiety fused to said βγΙΡ or said RET donor, RET acceptor or first fragment.

1 . The biosensor system of claim 16, wherein said P -targeting moiety is fused at the C- terminus of said RET donor, RET acceptor or first fragment.

18. The biosensor system of claim 16 or 17, wherein said PM-targeting moiety comprises a prenylation motif.

19. The biosensor system of claim 16, wherein said prenylation motif is the prenylation motif of human KRAS splice variant b.

20. The biosensor system of claim 19, wherein said PM-targeting moiety comprises the amino acid sequence K K KKSKTKCVIM (SEQ ID NO:37).

21 . The biosensor system of any one of claims 16 to 20, further comprising a flexible linker between (i) said RET donor, RET acceptor or first fragment and (ii) said P -targeting moiety.

22. The biosensor system of claim 21 , wherein said flexible linker has a length corresponding to about 50 to about 500 amino acids.

23. The biosensor system of claim 22, wherein said flexible linker has a length corresponding to about 200 amino acids.

24. The biosensor system of any one of claims 1 to 23, wherein said recombinant Ga protein is human Ga_q, Ga_s, Ga_n, Ga_i2, Ga_i3, Ga_t._cone, Ga_t._rod, Ga_t-_gust, Ga_z, Ga_oA, Ga_oB, Ga₀i_f, Ga-n , Ga-,2, Ga₁₃, Ga₁₄, and Ga₁₅/Ga₁₆ protein, or promiscuous or non-selective Ga variant thereof.

25. The biosensor system of any one of claims 1 to 24, wherein said βγΙΡ is GRK2 or GRK3.

26. The biosensor system of any one of claims 1 to 25, wherein (i) if said second component comprises a fused Gp protein, said first and second biosensors further comprises a recombinant Gy protein, or (ii) if said second component comprises a fused Gy protein, said first and second biosensors further comprises a recombinant Gp protein.

27. The biosensor system of any one of claims 1 to 25, wherein the biosensor system defined in (A) further comprises a G-protein-coupled receptor (GPCR).

28. The biosensor system of any one of claims 1 to 27, wherein the biosensor system defined in (B) further comprises a recombinant Gp protein and/or a recombinant Gy protein.

29. The biosensor system of any one of claims 1 to 28, wherein said first biosensor is present in a first cell and said second biosensor is present in a second cell.

30. The biosensor system of any one of claims 1 to 28, wherein in the biosensor system defined in (A), said first biosensor is present in a first membrane preparation and said second biosensor is present in a second membrane preparation.

31 . The biosensor system of any one of claims 1 to 30, wherein the biosensor system defined in (A) comprises a plurality of second biosensors, wherein each of said second biosensors comprises a different recombinant Ga protein.

32. The biosensor system of claim 31 , wherein said different recombinant Ga proteins are at least two of the following Ga proteins: Ga_q, Ga_s, Ga_n , Ga_i2, Ga_i3, Ga_t -cone, GOt-rod, GOt-gust, GO_z, Ga_oA, Ga_oB, Ga₀,_f, Gan, Ga₁₂, Ga₁₃, Ga₁₄, and Ga₁₅/Ga₁₆.

33. A nucleic acid comprising a sequence encoding the first, second and third components defined in any one of claims 1 to 26.

34. The nucleic acid of claim 33, further comprising a sequence encoding a Θγ protein or a Gp protein.

35. The nucleic acid of claim 33 or 34, further comprising one or more translation regulatory sequences.

36. The nucleic acid of claim 35, wherein said one or more translation regulatory sequences are Internal Ribosome Entry Site (IRES).

37. A biosensor for detecting G-protein activity comprising:

38. The biosensor of claim 37, wherein said PM targeting moiety is a PM protein or a fragment thereof that localizes to the PM.

39. The biosensor of claim 38, wherein said PM protein or fragment thereof comprises (a) a palmitoylation, myristoylation, and/or prenylation signal sequence and/or (b) a polybasic sequence.

40. The biosensor of claim 39, wherein said polybasic sequence and prenylation signal sequence are from human KRAS splice variant b.

41 . The biosensor of claim 40, wherein said PM targeting moiety comprises the amino acid sequence K K KKSKTKCVIM (SEQ ID NO:37).

42. The biosensor of any one of claims 37 to 41 , wherein said biosensor further comprises a third component that comprises a recombinant Ga protein.

43. The biosensor of claim 42, wherein said recombinant Ga protein is of the Gq family.

44. The biosensor of claim 43, wherein said recombinant Ga protein is Ga_q or Ga-n.

(1) providing a biosensor comprising the elements defined in (A), (B) or (C):

(A) (i) a first component comprising a Θβγ interacting protein (βγΙΡ) fused to (a) a RET donor; (b) a RET acceptor or (c) a first fragment of a reporter protein;

(ii) a second component comprising a fused GB protein or a fused Gy protein, wherein said Gp protein or said Gy protein is fused to (a) a RET donor; (b) a RET acceptor or (c) a second fragment of said reporter protein,

wherein (a) if said βγΙΡ is fused to said RET donor, said Gp or Gy protein is fused to said RET acceptor; (b) if said βγΙΡ is fused to said RET acceptor, said Gp or G protein is fused to said RET donor; and (c) if said βγΙΡ is fused to said first fragment of said reporter protein, said Gp or Gy protein is fused to said second fragment of said reporter protein;

(iii) a third component comprising a recombinant Ga protein; and

(iv) a fourth component comprising said GPCR;

(B)

(i) a first component comprising a Gpy interacting protein (βγΙ Ρ) fused to (a) a RET donor; (b) a RET acceptor or (c) a first fragment of a reporter protein;

(ii) a second component comprising said GPCR fused at its C-terminal to (a) a RET donor; (b) a RET acceptor or (c) a second fragment of said reporter protein;

(iii) a third component comprising a recombinant Ga protein;

wherein (a) if said βγΙΡ is fused to said RET donor, said GPCR is fused to said

RET acceptor; (b) if said βγΙΡ is fused to said RET acceptor, said GPCR is fused to said RET donor; and (c) if said βγΙΡ is fused to said first fragment of said reporter protein, said GPCR is fused to said second fragment of said reporter protein; or

(C)

(i) a first component comprising a Gpy interacting protein (βγΙΡ) fused to (a) a RET donor; (b) a RET acceptor or (c) a first fragment of a reporter protein;

wherein (a) if said βγΙΡ is fused to said RET donor, said PM-targeting moiety is fused to said RET acceptor; (b) if said βγΙΡ is fused to said RET acceptor, said PM-targeting moiety is fused to said RET donor; and (c) if said βγΙΡ is fused to said first fragment of said reporter protein, said PM-targeting moiety is fused to said second fragment of said reporter protein;

(iii) a third component comprising a recombinant Ga protein; and (iv) a fourth component comprising said GPCR; and

46. The method of claim 44, wherein said biosensors comprise one or more of the features defined in claims 2 to 32 and 38 to 44.

(a) measuring the signal emitted by said RET acceptor or reporter protein in the presence and absence of said GPCR agonist in the first and second biosensors of the biosensor system of any one of claims 1 to 32, and

(a) measuring the signal emitted by a RET acceptor or reporter protein in the presence and absence of said GPCR agonist in a first biosensor comprising:

(i) a first component comprising a ΰβγ interacting protein (βγΙΡ) fused to (a) a RET donor; (b) a RET acceptor or (c) a first fragment of a reporter protein; and

(i) the first and second components defined in (a); and

(ii) a third component comprising a recombinant form of said Ga protein; wherein (a) if said βγΙΡ is fused to said RET donor, said GPCR is fused to said RET acceptor; (b) if said βγΙΡ is fused to said RET acceptor, said GPCR is fused to said RET donor; and (c) if said βγΙΡ is fused to said first fragment of said reporter protein, said GPCR is fused to said second fragment of said reporter protein;

49. The method of claim 47, wherein said biosensors comprise one or more of the features defined in claims 38 to 44.

50. The method of claim 45, further comprising

(3) measuring the signal emitted by said RET acceptor or reporter protein in

(a) the second biosensor(s) defined in element (A) of any one of claims 1 to 31 , (b) the biosensor defined in element (B) of any one of claims 1 to 31 , or

(c) the biosensor of any one of claims 42 to 44,

(4) determining whether said test agent is an inhibitor of said Ga protein;

(1) contacting

(a) the second biosensor(s) defined in element (A) of any one of claims 1 to 32,

(b) the biosensor defined in element (B) of any one of claims 1 to 32, or

(c) the biosensor of any one of claims 42 to 44;

with a GPCR agonist, wherein said recombinant Ga protein corresponds to said Ga protein of interest;

(c) determining whether said test agent is an inhibitor of said Ga protein,

(1) contacting

(b) the biosensor defined in element (B) of any one of claims 1 to 32, or

(c) the biosensor of any one of claims 42 to 44;

(3) determining whether said test agent is an activator of said Ga protein,

53. The method of any one of claims 45 to 52, wherein said RET donor is a bioluminescent protein, and wherein said method further comprises contacting the biosensor with a substrate for said donor bioluminescent protein.

54. The method of claim 53, wherein said substrate is a luciferin.

55. The method of claim 54, wherein said luciferin is a coelenterazine.

56. The method of claim 55, wherein said coelenterazine is Coelenterazine 400A.

57. The method of any one of claims 45 to 56, wherein the biosensor comprises a RET donor and a RET acceptor, and wherein said method further comprises: (i) measuring signal emitted by said RET donor, and (ii) determining the ratio [RET acceptor signal / RET donor signal].

59. The mutated Ga polypeptide of claim 58, wherein said mutation is a substitution.

60. The mutated Ga polypeptide of claim 58 or 59, wherein said mutation is at a position corresponding to residue 67 of human Ga_q protein.

61 . The mutated Ga polypeptide of claim 60, wherein said mutation is a substitution for a non-aromatic residue.

62. The mutated Ga polypeptide of claim 61 , wherein non-aromatic residue is cysteine.

63. The mutated Ga polypeptide of claim 58 or 59, wherein said mutation is at a position corresponding to residue 75 of human Ga_q protein.

64. The mutated Ga polypeptide of claim 63, wherein said mutation is a substitution for a non-aromatic residue.

65. The mutated Ga polypeptide of claim 64, wherein said non-aromatic residue is glycine.

66. A nucleic acid comprising a sequence encoding the mutated Ga polypeptide of any one of claims 58 to 65.

67. A plasmid or vector comprising the nucleic acid of claim 66.

68. A cell comprising the nucleic acid of claim 65 or the plasmid of claim 67.