Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
  • G01N33/567—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds utilising isolate of tissue or organ as binding agent
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances

Definitions

• compositions and methods for monitoring the movement of analytes and/or cellular components across biological membranes e.g., cell surface internalization.
• reporter constructs are provided, the transmembrane movement of which (e.g., by endocytosis), is monitored by methods described herein.
• Endocytosis is a critical mechanism central to, among other processes, receptor desensitization and signal transduction.
• Available technologies for measuring endocytosis based upon high-content imaging are biologically predictive, but limited in terms of ease of use.
• high throughput screening (HTS)-compatible technologies such as those utilizing enzyme fragment complementation, require excessive engineering of the biological system, often leading to screening errors (e.g., false negative hits in HTS).
• the present invention provides methods of detecting endocytosis comprising: (a) tethering a reporter element to a cell-surface analyte to create a fusion element; and (b) monitoring a detectable signal from the reporter element thereby detecting endocytosis.
• the reporter element emits the detectable signal upon interaction with a reporter substrate.
• the method further comprises a step between steps (a) and (b) of contacting the reporter element with a reporter substrate.
• the method further comprises a step between steps (a) and (b) of allowing endocytosis of the cell-surface analyte, the reporter element, and/or the fusion element.
• the detectable signal from the reporter element is altered upon endocytosis of the reporter element.
• monitoring the detectable signal thereby detecting endocytosis comprises: (i) allowing endocytosis of the fusion element; and (ii) detecting the signal after induction of endocytosis.
• monitoring the detectable signal to detect endocytosis comprises: (i) detecting the signal from the reporter element prior to endocytosis; (ii) allowing endocytosis of the fusion element; (iii) repeating detection of the signal from the reporter element; and (iv) comparing the signal from step (i) to the signal from step (iii) to detect endocytosis.
• allowing endocytosis of the fusion element comprises allowing time (e.g., sufficient time) for endocytosis to occur (e.g., 1 sec...2 sec... 5 sec...30 sec... 1 min... 2 min... 5 min... 30 min... 60 min, or more).
• the cell-surface analyte is a cell surface component.
• the cell surface component comprises a cell surface receptor.
• the cell-surface analyte is non-covalently associated with a component of the cell surface.
• the reporter element and the cell-surface analyte are expressed as a fusion protein.
• the reporter element and the cell-surface analyte are covalently or non-covalently coupled (e.g., after translation). In some embodiments, the reporter element and the cell-surface analyte are expressed as a fusion protein. In some embodiments, the detectable signal comprises an optical signal. In some embodiments, the detectable signal comprises luminescence. In some embodiments, the reporter element comprises a luciferase enzyme. In some embodiments, the luciferase enzyme is a beetle or Oplophorus luciferase enzyme.
• a luciferase enzyme is a wild-type luciferase (e.g., beetle or Oplophorus luciferase enzyme) or a mutant thereof (e.g., >70% sequence identity to wild-type).
• endocytosis results in movement of the reporter element from the extracellular space to the intracellular space.
• the reporter element moves across a biological membrane (e.g., plasma membrane).
• the reporter element translocates into a new membrane microenvironment.
• the detectable signal is detectably altered (e.g., gain of signal, reduction of signal, change in emission property, etc.) within the intracellular space compared to the extracellular space.
• the detectable signal from the reporter is reduced within the intracellular space compared to the extracellular space.
• the intracellular space comprises an endosome.
• the detectable signal is detectably altered (e.g., gain of signal, reduction of signal, change in emission property, etc.) within the endosome compared to the extracellular space.
• the detectable signal from the reporter is reduced within the endosome compared to the outside of the cell.
• the method further comprises a step between steps (i) and (ii) of triggering endocytosis.
• endocytosis is triggered by one or more of: receptor agonists (e.g., small molecule, cytokines, etc.), modified growth conditions, pharmacologic compounds, changes in cellular signaling pathways, presence of any endocytosis inducing species, etc.
• the reporter substrate is permeable to the plasma membrane of live cells. In some embodiments, the reporter substrate is permeable to cellular membranes. In some embodiments, the reporter substrate is completely or largely excluded from the intracellular space of the cell. In some embodiments, the reporter is capable of turning over multiple reporter substrates (e.g., multiple substrate turnover).
• the reporter substrate is a luciferin, luciferin derivative, coelenterazine, or a coelenterazine derivative.
• the present invention provides methods of detecting endocytosis comprising: (a) incorporating a fusion element into the cell membrane of a cell, wherein the fusion element comprises a cell-surface analyte and a reporter element; (b) contacting the reporter with a reporter substrate, wherein the contacting produces a detectable signal; and (c) monitoring the detectable signal thereby detecting endocytosis.
• monitoring of the detectable signal comprises: (i) allowing endocytosis of the fusion element; and (ii) detecting the signal after induction of endocytosis.
• monitoring of the detectable signal comprises: (i) detecting the signal prior to endocytosis; (ii) allowing endocytosis of the fusion element; (iii) detecting the signal following endocytosis; and (iv) comparing the signal from step (i) to the signal from step (iii) to detect endocytosis.
• the fusion element comprises a fusion protein.
• allowing endocytosis comprises allowing time (e.g., sufficient time) for endocytosis to occur (e.g., 1 sec...2 sec... 5 sec...30 sec... 1 min... 2 min... 5 min... 30 min... 60 min, or more).
• the cell-surface analyte comprises a cell surface component. In some embodiments, the cell surface component comprises a cell surface receptor. In some embodiments, the cell-surface analyte is non-covalently associated with a component of the cell surface.
• the reporter element comprises a luciferase enzyme. In some embodiments, the luciferase enzyme is a beetle or Oplophorus luciferase enzyme. In some embodiments, the luciferase enzyme is a wild-type enzyme (e.g., beetle or Oplophorus luciferase enzyme) or a mutant thereof (e.g., >70% sequence identity).
• the fusion element is incorporated into the cell membrane such that the reporter element is localized in the extracellular space (e.g., adjacent to the cell).
• endocytosis of the reporter element results in movement of the reporter element from an extracellular space into an endosome.
• the signal is detectably altered within the endosome compared to the extracellular space.
• the signal is reduced within the endosome compared to the extracellular space.
• the method further comprises a step between steps (i) and (ii) of triggering endocytosis.
• endocytosis is triggered by one or more of: receptor agonists (e.g., small molecule, cytokines, etc.), modified growth conditions, pharmacologic compounds, changes in cellular signaling pathways, presence of any endocytosis inducing species, etc.
• the reporter substrate is known to be permeable to the plasma membrane of live cells.
• the reporter substrate is permeable to cellular membranes or is completely or largely excluded from the topological inside of the cell under the conditions of the experiment.
• the reporter substrate is a luciferin, luciferin derivative, coelenterazine or coelenterazine derivative.
• the present invention provides methods of detecting endocytosis comprising one or more of the steps of (e.g., all of the steps, all of the steps in order): (a) incorporating a fusion protein into the cell membrane of a cell, wherein the fusion protein comprises a reporter polypeptide and a cell surface receptor, wherein the reporter polypeptide emits a detectable signal upon interaction with a reporter substrate; (b) contacting the reporter polypeptide with the reporter substrate; (c) detecting the detectable signal; (d) inducing endocytosis and/or allowing endocytosis to occur; (e) repeating detection of the detectable signal; and (f) comparing the signal from step (c) to the signal from step (e), wherein alteration of the signal indicates that endocytosis occurred.
• the fusion protein is expressed from a fusion construct. In some embodiments, the fusion protein is generated based upon antibody-antigen association. In some embodiments, the fusion protein is expressed within the cell. In some embodiments, the cell-surface analyte is produced exogenously and added to the medium of the cell. In some embodiments, the reporter substrate is cell membrane permeable. In some embodiments, the reporter polypeptide is capable of multiple turnovers of the reporter substrate. In some embodiments, the reporter polypeptide comprises a luciferase enzyme. In some embodiments, the luciferase enzyme is a beetle or Oplophorus luciferase enzyme.
• the luciferase enzyme is a wild-type enzyme (e.g., beetle or Oplophorus luciferase enzyme) or a mutant thereof (e.g., >70% sequence identity).
• the fusion protein is incorporated into the cell membrane such that the reporter element is localized to the outside of the cell.
• endocytosis of the reporter polypeptide results in movement of the reporter polypeptide from an extracellular space into an intracellular space.
• the signal is detectably altered within the intracellular space compared to the extracellular space.
• the intracellular space into is an endosome.
• the signal is detectably altered within the endosome compared to the extracellular space.
• the signal is reduced within the intracellular space compared to the extracellular space. In some embodiments, the signal is reduced within the endosome compared to the extracellular space.
• the reporter substrate is known to be permeable to the plasma membrane of live cells. In some embodiments, the reporter substrate is permeable to cellular membranes or is completely or largely excluded from the topological inside of the cell under the conditions of the experiment. In some embodiments, the reporter substrate is a luciferin, luciferin derivative, coelenterazine or coelenterazine derivative.
• the present invention provides compositions and methods of detecting endocytosis comprising: (a) tethering a reporter element to a cell-surface analyte; and (b) monitoring a detectable signal from the reporter element thereby detecting
• the reporter element emits a detectable signal upon interaction with a reporter substrate.
• the method further comprises a step between steps (a) and (b) of contacting the reporter element with a reporter substrate.
• the method further comprises a step between steps (a) and (b) of allowing time for the endocytosis of the cell-surface analyte and/or the reporter element.
• the detectable signal from the reporter element is altered upon
• monitoring the detectable signal to detect endocytosis comprises: (i) allowing endocytosis of the fusion element and (ii) detecting the signal induction of endocytosis. In some embodiments, monitoring the detectable signal to detect endocytosis comprises: (i) detecting the signal prior to endocytosis; (ii) allowing endocytosis of the fusion element; (iii) detecting the signal following
• the reporter element is tethered to a cell surface component.
• the cell surface component comprises a cell surface receptor.
• a cell surface receptor comprises a sequence of SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, or SEQ ID NO. 10.
• a cell surface receptor comprises greater that 70% sequence identity (e.g., 75%... 80%... 90%... 95%... 98%... 99%) with one of SEQ ID NOS. 5-10.
• a cell surface receptor retains all or a portion of the biological activity of one of SEQ ID NOS. 5-10.
• the reporter element and the cell surface component are expressed as a fusion protein.
• a fusion protein comprises one or more peptide elements in addition to the reporter element and the cell surface component.
• a fusion protein comprises one or more signal peptides (e.g., IL6 signal peptide).
• a fusion protein comprises a signal peptide of 70% or greater sequence identity (e.g., 70%... 80%... 90%... 95%... 98%... 99%) with SEQ ID NO. 1.
• a fusion protein comprises one or more linker segments (e.g., GSSG linker).
• a linker segment is a peptide or non- peptide (e.g. nucleic acid, PEG, alkyl chain) segment connecting two polypeptide portions of the fusion protein.
• a fusion protein comprises a linker of 70% or greater sequence identity (e.g., 70%... 80%... 90%... 95%... 98%... 99%) with SEQ ID NO. 2.
• the detectable signal comprises an optical signal.
• the detectable signal comprises luminescence.
• the reporter element comprises a luciferase enzyme.
• the luciferase enzyme comprises a sequence of SEQ ID NO. 3 or SEQ ID NO. 4. In some embodiments, the luciferase enzyme comprises greater that 70%> sequence identity (e.g., 75%... 80%>...
• the luciferase enzyme retains all or a portion of the biological activity of one of SEQ ID NOS. 3- 4.
• endocytosis results in movement of the reporter element from the outside of the cell into an endosome. In some embodiments, endocytosis results in movement of the reporter element from the extracellular space into an intracellular space (e.g., an endosome).
• the detectable signal is detectably altered within the endosome compared to the outside of the cell. In some embodiments, the detectable signal from the reporter element is reduced within the endosome compared to the outside of the cell.
• the method further comprises a step between steps (i) and (ii) of triggering endocytosis (e.g., with a chemical (e.g., with a pharmaceutical agent)).
• the reporter substrate is added to the cell medium.
• the reporter substrate is permeable to the plasma membrane of live cells, is permeable to cell membranes, or is completely or largely excluded from the topological inside of the cell.
• the present invention provides methods of detecting endocytosis comprising: (a) incorporating a fusion element into the cell membrane of a cell, wherein the fusion element comprises an cell-surface analyte and a reporter element; (b) contacting the reporter element with a reporter substrate, wherein the contacting produces a signal from the reporter element; and (c) monitoring the signal thereby detecting endocytosis.
• monitoring the signal to detect endocytosis comprises: (i) detecting the signal prior to endocytosis; (ii) allowing endocytosis of the fusion element and cell- surface analyte; (iii) detecting the signal following endocytosis; and (iv) comparing the signal from step (i) to the signal from step (iii) thereby detecting endocytosis.
• the fusion element comprises a fusion protein.
• the cell- surface analyte comprises a cell surface component.
• the cell surface component comprises a cell surface receptor.
• the reporter element comprises a luciferase enzyme (e.g., beetle or Oplophorus luciferase enzyme).
• the luciferase enzyme is a wild-type enzyme (e.g., beetle or Oplophorus luciferase enzyme) or a mutant thereof (e.g., >70% sequence identity).
• the fusion element is incorporated into the cell membrane such that the reporter element is localized to the outside of the cell.
• endocytosis of the reporter element results in movement of the reporter element from the outside the cell into an endosome.
• the signal is detectably altered within the endosome compared to the outside of the cell.
• the signal is reduced within the endosome compared to the outside of the cell.
• the emission property, e.g., wavelength, of the signal is altered within the endosome compared to outside the cell.
• the method further comprises a step between steps (i) and (ii) of triggering endocytosis.
• the reporter substrate is permeable to the plasma membrane of live cells, is permeable to cell membranes, or is completely or largely excluded from the topological inside of the cell.
• the reporter substrate is a membrane-permeable substrate.
• the present invention provides methods of detecting endocytosis comprising: (a) incorporating a fusion element into the cell membrane of a cell, wherein the fusion element comprises an cell-surface analyte and a reporter element; (b) allowing endocytosis to occur and/or inducing endocytosis; (c) contacting the reporter element with a reporter substrate, wherein the contacting produces a signal from the reporter element; and (d) monitoring the signal thereby detecting endocytosis.
• the present invention provides methods of detecting endocytosis comprising: (a) incorporating a fusion protein into the cell membrane of a cell, wherein the fusion protein comprises a reporter polypeptide and a cell-surface receptor, wherein the reporter polypeptide emits a detectable signal upon interaction with a reporter substrate; (b) contacting the reporter polypeptide with the reporter substrate; (c) detecting the detectable signal from the reporter polypeptide; (d) inducing endocytosis and/or allowing endocytosis to occur; (e) repeating detection of the detectable signal; and (f) comparing the signal from step (c) to the signal from step (e), wherein alteration of the signal indicates that endocytosis occurred.
• the fusion protein is expressed from a fusion construct.
• the fusion protein is expressed endogenously within the cell.
• the fusion protein is expressed exogenously and added to the cell medium.
• the present invention provides fusion proteins comprising a reporter protein and cell surface component, and methods of use thereof, e.g., for detecting endocytosis.
• a reporter protein is a reporter enzyme.
• the reporter enzyme is a luciferase.
• the luciferase is a firefly luciferase, Oplophorus luciferase, Renilla luciferase, variants or mutants thereof, or other suitable luciferase.
• the luciferase has 70% or greater sequence identity to all or a portion of SEQ ID NO. 3 or SEQ ID NO. 4.
• the luciferase has the biological activity of a luciferase of SEQ ID NO. 3, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, or SEQ ID NO. 10. In some embodiments, the luciferase has the biological activity of a luciferase of SEQ ID NO. 3 or SEQ ID NO. 4.
• a cell surface component is a cell surface protein. In some embodiments, the cell surface protein is a cell surface receptor. In some embodiments, the cell surface receptor has 70%> or greater sequence identity to all or a portion of SEQ ID NO. 3, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO.
• a fusion protein comprises one or more signal peptides (e.g., cell surface signals, secretion signals, etc.).
• the signal peptide has 70%> or greater sequence identity to all or a portion of SEQ ID NO. 1.
• the signal peptide has the biological activity of a signal peptide of SEQ ID NO. 1.
• a fusion protein comprises one or more linker segments.
• the linker segment is a peptide or non-peptide. In some embodiments, the linker segment is a polymer. In some embodiments, the linker segment is a carbon- containing chain. In some embodiments, the linker is a GSSG linker. In some embodiments, the linker comprises 70% or greater sequence identity to all or a portion of SEQ ID NO. 2. In some embodiments, the linker covalently connects a cell surface component, reporter, and/or signal peptide with one or more of a cell surface component, reporter element, and/or signal peptide. In some embodiments, a secretion tag (e.g., IL6 secretion tag (e.g., SEQ ID NO.
• a secretion tag e.g., IL6 secretion tag (e.g., SEQ ID NO.
• linker e.g., GSSG linker (e.g., SEQ ID NO. 2)
• linker e.g., GSSG linker (e.g., SEQ ID NO. 2)
• the secretion tag is attached to the N-terminus of a luciferase reporter enzyme by a linker.
• the C-terminal end of a luciferase reporter enzyme is attached to the N-terminal end of a cell surface component, e.g., a cell surface protein or receptor, either directly or by a linker.
• the fusion protein comprises the following formula:
• the fusion protein comprises the following formula:
• L is an optional linker segment.
• the fusion protein comprises the following formula:
• the fusion protein comprises the following formula:
• the fusion protein comprises the following formula:
• the fusion protein comprises the following formula:
• L is an optional linker segment.
• the fusion protein comprises the following formula: N-Signal Peptide - Cell Surface Protein - Reporter-C;
• Li is an optional linker segment
• L 2 is a second optional linker segment
• the fusion protein comprises the following formula:
• kits comprising components, reagents (e.g., fusion constructs, reporter proteins, substrates, etc.), materials (e.g., solid supports (e.g., multiwell plates), cells), and/or systems/devices (e.g., fluorometers, cell imaging, data analysis, etc.), for performing assays described herein.
• a kit of the present invention may comprise a fusion construct comprising a reporter protein, e.g., a luciferase enzyme, and a substrate for the reporter protein, e.g., a luciferin or coelenterazine or derivatives thereof.
• the present invention provides methods of detecting transmembrane trafficking comprising: (a) incorporating a fusion protein into a membrane of a cell, wherein the fusion protein comprises a reporter element and a membrane-associated element, wherein the reporter element emits a detectable signal upon interaction with a reporter substrate; (b) contacting the reporter element with the reporter substrate; and (c) detecting the detectable signal.
• the method further comprises the steps of: (d) inducing transmembrane trafficking and/or allowing transmembrane trafficking to occur; (e) repeating detection of the detectable signal; and (f) comparing the signal from step (c) to the signal from step (e), wherein alteration of the signal indicates that
• the method further comprises a step between steps (a) and (b) inducing endocytosis and/or allowing transmembrane trafficking to occur. In some embodiments, the method further comprises: (f) comparing the signal from step (c) to a control signal, wherein alteration of the signal indicates that transmembrane trafficking occurred.
• the reporter element is an enzyme. In some embodiments, the signal is an optical signal. In some embodiments, the reporter substrate is membrane permeable. In some embodiments, the membrane-associated element is a membrane-associate polypeptide.
• Figure 1 shows graphs depicting kinetic measurements of the endocytosis of fusion proteins of GPCRs fused to firefly luciferase in response to agonist or vehicle-only: (a) Firefly-Luc/ATIR internalization, (b) Firefly- Luc/OPRDl internalization, (c) Firefly- Luc/B2AR internalization, and (d) Firefly-Luc /V2R internalization.
• Figure 2 shows fixed time-point concentration response curves: (a) Normalized concentration-response curves for 4 different GPCRs using a thermostable firefly luciferase (FFluc). The luminescence from each sample (agonist concentration) at 20 minutes was normalized to the luminescence of the same sample prior to stimulation; (b) EGF
• Figure 3 shows single dose, normalized kinetic responses for endocytosis of various OgLuc/GPCR fusions in real time.
• Figure 4 shows normalized GPCR concentration-response curves for endocytosis of various OgLuc/GPCR fusions.
• luminescence at 30 minutes was normalized to the luminescence of the same sample prior to stimulation.
• the normalized luminescence was plotted against agonist concentration
• Figure 5 shows graphs demonstrating agonist specificity of endocytosis induction of B2AR and V2R using OgLuc reporter proteins using methods as described above;
• (c-d) shows concentration response curves for endocytosis of OgLuc B2AR fusions using alternate coelenterazine derivatives as substrates (PBI-4525 and PBI-4377) as described above;
• (e-f) demonstrates the ability to measure endocytosis of OgLuc-B2AR or OgLuc- V2R by adding luciferase substrate after endocytosis has occurred (nonkinetic, endpoint measurement).
• Figure 6 shows an isoproterenol concentration response curve for OgLuc/B2AR endocytosis in the presence or absence of a B2AR endocytosis antagonist , alperenerol.
• Figure 7 shows a concentration response curve for FFLuc/B2AR endocytosis in the presence of co-expressed dominant-negative dynamin, demonstrating dependence on functional dynamin proteins for this assay readout
• Figure 8 shows a graph demonstrating G-protein independent signaling events using known ⁇ -arrestin-biased agonist TRV120027 with angiotensin-typel A receptor/firefly luciferase fusions.
• Figure 9 shows the chemical structures of examples of luciferase substrates.
• transmembrane trafficking refers to the movement of an analyte, cell surface component, protein, etc. across a biological membrane or lipid bilayer, by any suitable mechanism.
• transmembrane trafficking applies to movement across the cell membrane, intracellular membranes, vesicular membranes, the nuclear membrane, an organelle membrane (e.g., chloroplast, mitochondria, endoplasmic reticulum, golgi complex, endosomes, vacuoles, etc.).
• transmembrane trafficking applies to movement across a membrane by any suitable mechanism (e.g., endocytosis, phagocytosis, autophagic flux, membrane translocation, exocytosis, autophagy, transmembrane movement, etc.).
• extracellular refers a physical space not contained within a cell, outside a cell, outside of a cell membrane, and/or not contained within a cell membrane.
• extracellular refers a physical space not contained within a cell, outside a cell, outside of a cell membrane, and/or not contained within a cell membrane.
• the region immediately adjacent to a cell, but not within the plasma membrane is defined as being extracellular.
• intracellular As used herein, the terms “intracellular,” “intracellular space,” and “intracellular region” refer a physical space contained within a cell and/or enveloped within a cell membrane.
• the "cytoplasmic,” “nuclear,” “endosomal,” and other compartments or organelles within a cell are within the “intracellular space.”
• compositions and methods for monitoring the movement of analytes and/or cellular components across biological membranes e.g., cell surface internalization.
• reporter constructs are provided, the transmembrane movement of which (e.g., by endocytosis), is monitored by methods described herein.
• compositions and methods described herein are suitable for the detection and monitoring of various types of transmembrane trafficking. However, most embodiments described herein specifically focus on cell surface internalization and/or endocytosis. It should be noted that, in some embodiments, the compositions and methods described herein are suitable for detecting and/or monitoring other types of trafficking (e.g., phagocytosis, autophagic flux, membrane translocation, exocytosis) across other cellular membranes (e.g., intracellular membranes, vesicular membranes, the nuclear membrane, organelle membranes, etc.).
• trafficking e.g., phagocytosis, autophagic flux, membrane translocation, exocytosis
• other cellular membranes e.g., intracellular membranes, vesicular membranes, the nuclear membrane, organelle membranes, etc.
• Embodiments described herein are not limited to any one type of membrane trafficking or any one class of membranes. Embodiments herein that specifically address endocytosis should be viewed as more broadly applying to other type of transmembrane trafficking as well.
• compositions and methods for monitoring cell-surface internalization and/or endocytosis by cells are provided herein.
• reporter constructs e.g., fusions of cell surface components and reporter proteins
• the internalization of which e.g., by endocytosis
• compositions e.g., cell surface component/reporter fusion proteins
• methods of use thereof that provide a simple method to monitor endocytosis in real time, in living cells, and/or without the disadvantages of the existing methods are provided.
• methods are performed on a solid support, e.g., microplate.
• methods and compositions are provided for determining the amount, rate, duration, timing, triggers, and/or inhibitors of endocytosis of a cell surface component, e.g., cell surface receptor or protein.
• the internalization assay methods described herein are minimally invasive, compatible with transiently transfected cells, and/or utilize a non-destructive endpoint for multiplex analysis of endocytosis with other functional assay endpoints, e.g., measurements of second messenger signaling, viability, cytotoxicity or similar phenotypic assays.
• compositions and methods for monitoring and/or detecting the internalization and/or endocytosis of cell surface proteins e.g., a cell surface receptor, cell- surface analyte, etc.
• a reporter element e.g., a reporter protein (e.g., luciferase) that produces a detectable signal (e.g., through interaction with a substrate, via a detectable label, through an interaction with another molecule, etc.) is tethered to the cell surface in the non-cytoplasmic space (e.g., extracellular space, e.g., to a cell surface receptor).
• internalization or endocytosis of the region of the cell membrane or the particular cell membrane component to which the reporter element is attached results in internalization or endocytosis of the reporter element.
• a detectable change in the signal emitted e.g., loss of signal, reduction in signal, change of frequency, increase in signal, gain of signal, change in wavelength, etc.
• monitoring the signal emitted e.g., by endpoint detection, by real-time monitoring, etc. provides a method of detecting internalization of the reporter element.
• monitoring the signal emitted, and/or the internalization of the reporter molecule provides methods for studying or monitoring endocytosis (e.g., rate, timing, duration, amount, etc.) and the affects of various intracellular conditions, extracellular conditions, molecular components, and pharmaceutical agents thereon.
• endocytosis e.g., rate, timing, duration, amount, etc.
• compositions and methods for tethering a reporter element(s) to the cell surface detecting the signal emitted (e.g., on the cell surface or within a cell or endosome), monitoring reporter signals (e.g., in real time, via endpoint detection, etc.), assessing the affects of conditional changes (e.g., introduction of drug-like molecules) on endocytosis, etc., are provided.
• a reporter protein e.g., an enzyme (e.g., luciferase) is attached to a cell surface receptor, thereby tethering the reporter protein to the cell surface to monitor internalization and/or endocytosis of the cell surface receptor.
• a reporter protein and cell surface receptor protein are expressed as a single fusion protein, e.g., from a fusion construct (e.g., genetic construct).
• a reporter protein and cell surface receptor protein stably associate (e.g., covalently or non-covalently) to form a fusion.
• a reporter protein and cell surface receptor protein are non- genetically coupled to form a fusion protein.
• internalization e.g., through endocytosis
• the signal from the reporter protein is altered upon endocytosis of the cell surface receptor (and reporter).
• monitoring the signal from the reporter molecule over time provides a means for detecting internalization and/or endocytosis of the cell surface receptor (e.g., as the signal is altered upon
• a means of attaching, adhering, anchoring, associating, tethering, etc. a reporter element to the cell membrane e.g., to monitor internalization of the reporter and adjacent cell membrane
• a cell surface component is utilized to attach, adhere, anchor, associate, tether, etc. a reporter element to the cell surface.
• a fusion element comprising a reporter element and a cell surface component are provided.
• the cell surface component is any element, e.g., protein, capable of attaching, adhering, anchoring, associating, tethering, etc. a fusion element to a cell surface.
• the cell surface component comprises a protein, peptide, polypeptide, lipid, carbohydrate, viral particle, macromolecular complex, etc.
• the cell surface component is a protein, peptide, or polypeptide.
• the cell surface component is a membrane-associated protein, membrane -bound protein, cell surface receptor, transmembrane receptor, etc.
• a receptor is an ion channel-linked receptor (e.g., acetylcholine receptor), enzyme-linked receptor (e.g., receptor tyrosine kinases; tyrosine kinase associated receptors; receptor-like tyrosine phosphatases; receptor serine/threonine kinases; receptor guanylyl cyclases, histidine kinase associated receptors), and/or G-protein-coupled receptor/7- transmembrane receptor.
• enzyme-linked receptor e.g., receptor tyrosine kinases; tyrosine kinase associated receptors; receptor-like tyrosine phosphatases; receptor serine/threonine kinases; receptor guanylyl cyclases, histidine kinase associated receptors
• G-protein-coupled receptor/7- transmembrane receptor e.g., G-protein-coupled receptor/7- transmembrane receptor
• transmembrane receptors that find use in embodiments described herein include, but are not limited to: angiotensin2-type la receptor (AT1R), vasopressin 2 receptor (V2R), delta-opioid receptor (OPRD1) and epidermal growth factor receptor (EGFR) delta opioid receptor, vasopressin 2 receptor, EDGl receptor, p2-adrenergic receptor (ADRB2), arginine vasopressin receptor 2 (AVPR2), serotonin receptor la
• AT1R angiotensin2-type la receptor
• V2R vasopressin 2 receptor
• OPRD1 delta-opioid receptor
• EGFR epidermal growth factor receptor
• EGFR epidermal growth factor receptor
• vasopressin 2 receptor EDGl receptor
• ADRB2 p2-adrenergic receptor
• AVPR2 arginine vasopressin receptor 2
• HTRl A m2 muscarinic acetylcholine receptor (CHRM2), chemokine (C-C motif) receptor 5 (CCR5), dopamine D2 receptor (DRD2), kappa opioid receptor (OPR ), or ala-adregenic receptor (ADRAIA), the insulin growth factor- 1 receptor (IGF-R), etc.
• CHRM2 m2 muscarinic acetylcholine receptor
• CCR5 chemokine receptor 5
• D2 receptor dopamine D2 receptor
• OCR kappa opioid receptor
• ADRAIA ala-adregenic receptor
• IGF-R insulin growth factor- 1 receptor
• reporter elements or complexes are provided that emit a detectable signal that is altered (e.g., reduced) upon cellular internalization and/or
• a fusion element comprising a cell surface component and a reporter element
• the reporter element is any molecule, macromolecule (e.g., protein), or complex that produces a detectable signal, e.g., upon interaction with a substrate or upon stimulation (e.g., by change in pH, by exposure to light).
• a reporter element is a protein.
• a reporter element is an enzyme.
• a reporter, a reporter substrate, and/or a reporter/substrate complex is detectable by optical, spectroscopic, photochemical, biochemical, immunological, chemical and/or magnetic means.
• a reporter, a reporter substrate, and/or a reporter/substrate complex comprises a label.
• Suitable labels include, but are not limited to, colored, radioactive, fluorescent, ultraviolet, and/or magnetic molecules or particles.
• a reporter, a reporter substrate, and/or a reporter/substrate complex is labeled by one or more of:
• a reporter e.g., fluorescent, radioactive, optically detectable, contrast agent, etc. contacts, interacts, associates with, and/or binds a reporter.
• the substrate of a reporter is detectable.
• a reporter is detectable.
• a reporter is a peptide, polypeptide, or protein.
• reporter proteins include enzymes such as chloramphenicol acetyl transferase (CAT), ⁇ -glucuronidase (GUS) or ⁇ -galactosidase.
• CAT chloramphenicol acetyl transferase
• GUS ⁇ -glucuronidase
• a reporter is luminescent, fluorescent, and/or chemiluminescent.
• a reporter comprises proteins such as luciferases, beta lactamase, and alkaline phosphatase.
• a reporter comprises a luciferase, e.g., a beetle luciferase, e.g., a firefly luciferase (e.g., Photinus pyralis or Photuris pennsylvanica), Renilla luciferase, Gaussia luciferase, Oplophorus luciferase, luciferin-utilizing luciferases, coelenterazine-utilizing luciferases, and any suitable variants or mutants thereof.
• reporter proteins comprise enzymes capable of multiple substrate turnovers.
• reporter proteins are not limited to traditional enzymes.
• reporter proteins are wild-type proteins or mutated, e.g., thermostable and/or chemostable, forms which provide advantages over wild-type for use in reporter assays.
• a substrate, ligand, binding partner, label, etc. for a reporter element is provided.
• any suitable interaction partner (e.g., substrate, ligand, antibody, label, etc.) for a reporter element is provided.
• a reporter element is an enzyme, and an enzyme substrate is provided.
• the enzyme substrate is coelenterazine, luciferin, ⁇ other suitable luciferase substrates or derivatives thereof.
• interaction of a reporter enzyme and enzyme substrate provides a detectable signal, e.g., luminescence, from the enzyme interacting with the enzyme substrate.
• an interaction partner for a reporter element comprises a detectable label that is associated with the reporter upon interaction with the interaction partner.
• a substrate, ligand, binding partner, etc. is membrane permeable.
• a substrate, ligand, binding partner, etc. is capable or permeably passing through a cell membrane, endosomal membrane, vacuolar membrane, nuclear membrane, and/or other intracellular membrane.
• a substrate, ligand, binding partner, etc. is membrane impermeable.
• a substrate, ligand, binding partner, etc. is soluble in an intracellular, extracellular, and/or aqueous environment.
• a substrate for a reporter element e.g., enzyme
• a reporter element e.g., enzyme
• the present invention provides fusion proteins comprising one or more reporter elements, e.g., reporter proteins or enzymes (e.g., luciferase) attached either directly, by a linker, or through another element, to a cell surface component or integral cell membrane protein.
• a fusion protein comprises one or more additional peptide segments to provide functionality, e.g., cell export, secretion, or surface localization, or enhance the performance of the fusion protein.
• a fusion protein comprises a secretion peptide, e.g., IL-6 signal peptide (e.g., SEQ ID NO. 1).
• a fusion protein comprises an N-terminal secretion peptide, e.g., IL-6 signal peptide (e.g., SEQ ID NO. 1).
• a reporter element e.g., reporter enzyme
• a cell surface analyte e.g., cell surface protein
• a reporter element and cell surface analyte are genetically expressed as a fusion.
• a reporter element and cell surface analyte react (e.g., post-translationally) to form a fusion.
• a reporter element and cell surface analyte are coupled, linked, and/or attached covalently or non-covalently.
• the present invention provides fusion proteins comprising one or more reporter proteins, e.g., reporter enzyme (e.g., luciferase), covalently linked by a linker, e.g., GSSG linker (e.g., SEQ ID NO. 2), to a cell surface component or integral cell membrane protein.
• a fusion protein comprises one or more additional peptide segments attached to the reporter protein and/or cell surface component or integral cell membrane protein via a linker, e.g., GSSG linker (e.g., SEQ ID NO. 2).
• a linker is a peptide.
• suitable linkers provide adequate spacing between elements without interfering with the structure and/or biological activity of the elements.
• a linker is a non-peptide linker, e.g., polymer, alkyl chain, etc.
• suitable linkers are understood in the art. The present invention is not limited by the sequence of a peptide linker sequence.
• a reporter element e.g., a reporter protein or enzyme
• a reporter element is attached to the N-terminus, and/or an internal portion of a cell surface component, e.g., a cell surface receptor or other cell surface protein.
• a reporter element e.g., a reporter protein or enzyme
• a reporter element is attached to a portion of cell surface component so as to not interfere with the overall structure and/or function of the cell surface component.
• a cell surface receptor is attached to the N-terminus and/or an internal portion of a reporter element, e.g., a reporter protein or enzyme.
• a reporter element is attached to portion of cell surface component so as to not interfere with the overall structure and/or function of the reporter element.
• a reporter element is attached to the N-terminus of a cell surface receptor, e.g., GPCR, non-GPCR receptor (e.g., tyrosine kinase receptor, etc.).
• assays for the detection of endocytosis and reagents e.g., fusion proteins, reporter elements, cell surface components (e.g., cell surface receptors), substrates, expression constructs, test reagents, etc., materials (e.g., microplates) and devices (e.g., luminometers) for execution thereof are provided.
• the assays described herein exploit changes in the detectable signal emitted upon endocytosis.
• any reporter element e.g., reporter protein or enzyme (e.g., luciferase)) that emits a signal that is detectably different when tethered to the surface of a cell (e.g. by attachment to a cell surface protein) and following endocytosis finds use in the embodiments herein.
• reporter protein or enzyme e.g., luciferase
• the detectable change in the reporter signal upon endocytosis of the reporter element comprises: loss of signal, reduction of signal, enhancement of signal, gain of signal, alteration of signal (e.g., change in wavelength emitted, etc.), etc.
• the change in signal is due to the endocytic environment (e.g. H, salt concentration, steric constraint, etc.), degradation of the reporter element, recycling of reporter element to the cell surface, loss of substrate availability, or any other mechanism that results in a detectably different signal upon endocytosis of the reporter element.
• the present invention is not limited to any particular mechanism of altering the detectable signal of the reporter molecule, and an understanding of the mechanism of action is not necessary to practice the present invention.
• real-time detection and/or monitoring of endocytosis is provided.
• end- point e.g., detection and/or monitoring of endocytosis is provided.
• end-point e.g., detection and/or monitoring of endocytosis is compared to a control or standard to identify the presence, absence, rate, amount, etc. of endocytosis.
• the reporter signal is detected prior to addition of substrate.
• the reporter signal is detected following addition of substrate.
• the reporter substrate is added to cells prior to triggering of endocytosis.
• the reporter substrate is added to cells after triggering of endocytosis.
• the reporter substrate is added to cells prior to allowing endocytosis to occur.
• the reporter substrate is added to cells after allowing endocytosis to occur.
• the reporter signal and/or reporter/substrate signal is detected prior to endocytosis, e.g., to establish background, baseline, or pre-endocytosis level signal).
• time is allowed for endocytosis to occur.
• endocytosis is triggered by any suitable method, e.g., receptor agonists (e.g., small molecule, cytokines, etc.), modified growth conditions, pharmacologic compounds, changes in cellular signaling pathways, presence of any endocytosis inducing species, etc.
• a biological process that is known to lead to endocytosis is induced by any suitable method.
• the reporter signal and/or reporter/substrate signal is continuously or periodically (e.g., intervals of: Is, 2s, 5s, 10s, 30s, lm, 2m, 5, 10m, etc.) monitored following initial detection.
• the reporter signal and/or reporter/substrate signal is continuously or periodically monitored following addition of the reporter substrate.
• the reporter signal and/or reporter/substrate signal is continuously or periodically monitored following triggering of endocytosis, e.g., by addition of a receptor agonist.
• real-time monitoring of endocytosis is provided by detecting the reporter signal over time, e.g., at closely spaced intervals (e.g., Is, 2s, 5s, 10s, 30s, lm, 2m, etc.
• detection of endocytosis is provided by detecting the reporter signal at one or more fixed time points, e.g., 15 min, 20 min, 30 min., etc.
• measurement of the rate, amount, duration, etc. are provided by real-time assays.
• measurement of endocytosis is provided by detecting the reporter signal after stimulation of endocytosis via endpoint.
• compositions and methods for monitoring, detecting, or quantitating endocytosis are provided.
• compositions and methods described herein are useful for monitoring any type of endocytosis, e.g., clathrin-mediated, caveole, macropinocytosis, or phagocytosis.
• endocytosis is monitored irrespective of the endocytosis pathway and/or endocytic components involved in the process.
• compositions and methods are configured to detect all types of endocytosis.
• compositions and methods to detect dynamin-dependent endocytosis are provided.
• compositions and methods are provided to detect one or more types endocytosis, e.g., clathrin-mediated, caveole, macropinocytosis, phagocytosis, and/or dynamin-dependent.
• compositions and methods are provided to detect a single type of endocytosis, e.g., clathrin-mediated, caveole, macropinocytosis, dynamin-dependent, or phagocytosis.
• nucleic acid constructs and vectors are provided that encode and/or are capable of expressing one or more protein components (e.g., reporter, receptor, fusion, etc.) of the methods and assays described herein.
• vectors and/or constructs comprise sequences and regulatory elements that allow the encoded proteins to be expressed by the cellular transcription and translation machinery.
• vectors and/or constructs comprise one or more of enhancer regions, promoter regions, start codons, termination codons, transcription termination sequences, portable translation initiation sequences, gene coding regions, gene insertion regions, etc.
• constructs are provided that provide a cloning site for inserting a nucleic acid encoding a cell surface receptor, or other cell surface protein, for fusion with a reporter element.
• vectors are provided for stable or transient expression of reporter elements and/or fusion proteins in cells.
• suitable vectors, delivery systems e.g., viral gene delivery
• constructs are provided for producing the protein compositions and performing the methods described herein are provided.
• kits containing components, reagents, e.g., fusion constructs, reporter proteins, substrates, etc., and/or materials are provided.
• kits are provided for performing the methods or assays described herein.
• materials and reagents are provided in kits to produce, express, and/or engineer constructs, fusion proteins, etc. for carrying out methods of the present invention.
• methods and assays that are performed in a multiplex format are provided.
• methods and assays are performed in a high throughput manner, e.g., to screen inhibitors or enhancers of endocytosis.
• assays provided herein are rapidly performed in a multiwell plate, e.g., 96-well, 384-well, etc.
• assays measure endocytosis in high-throughput screening compatible, mix and read format (e.g., non-image based, flow-based, etc.).
• systems, devices, or apparatuses for assessing, quantitating, detecting, and/or monitoring the compositions, methods, and/or assays are provided. In some embodiments, systems, devices, and/or apparatuses are provided to detect, quantitate, or monitor, the amount of a reporter element on the exterior of a cell, the amount of reporter element endocytosed, and/or the endocytosis of reporter element.
• detection, quantification, and/or monitoring are provided by a device, system or apparatus comprising one or more of a spectrophotometer, fluorometer, luminometer, photomultiplier tube, photodiode, nephlometer, photon counter, electrodes, ammeter, scintillation counter, Geiger counter, voltmeter, capacitative sensors, radio-frequency transmitter,
• a spectrophotometer fluorometer, luminometer, photomultiplier tube, photodiode, nephlometer, photon counter, electrodes, ammeter, scintillation counter, Geiger counter, voltmeter, capacitative sensors, radio-frequency transmitter,
• magnetoresistometer flow cytometer, CCD, Hall-effect device, etc.
• a device suitable for detection of a given reporter element e.g., luciferase, beta lactamase, or radiolabel, is selected and/or provided.
• assays, methods, and compositions that are compatible detection systems that are comparatively inexpensive, e.g., low-moderate price luminometer as opposed to high-end fluorometer, are provided.
• methods to study or analyze endocytosis, and the role of intracellular or extracellular processes, components (e.g., clathrin, dynamin, etc.), and other factors (e.g., cell type, cell stress, extracellular environment, etc.) on receptor internalization and/or endocytosis are provided.
• assays are provided for monitoring the effect of various factors on the rate, amount, duration, and/or timing of receptor internalization and/or endocytosis.
• assays are provided for monitoring the effect of specific molecules, e.g., cellular components (e.g., clathrin, dynamin, etc.), drugs, etc., on the rate, amount, duration, and/or timing of receptor internalization and/or endocytosis.
• assays are provided (e.g., high throughput, multiplex, etc.) for screening compounds for a desired effect on internalization and/or endocytosis of a specific receptor.
• assays are provided for screening compounds to treat or prevent one or more conditions, diseases, or disorders that involve endocytosis, receptor internalization, or malfunction of a cell surface receptor.
• assays are provided for identifying molecules that trigger, enhance, inhibit, or otherwise affect the rate, amount, duration, and/or timing of the internalization of a specific receptor or endocytosis in general.
• GPCRs G-protein coupled receptors
• AT1R angiotensin2-type la receptor
• B2AR beta2-adrenergic receptor
• V2R vasopressin 2 receptor
• OPRDl delta-opioid receptor
• EGFR epidermal growth factor receptor
• HEK293 cells were transfected with plasmid DNA encoding the FFluc- GPCR fusions using Fugene HD (Promega Corp.) according to the manufacturer's instructions. To achieve proper expression levels, 1 part plasmid DNA (by mass) was diluted into 100 parts carrier DNA (pGEM-3Z; Promega Corp.) yielding approximately 0.5ng/well reporter DNA.
• plasmid DNA was transfected at 5ng/well and 0.16ng/well respectively.
• plasmid DNA was diluted 1 : 10 into carrier DNA yielding 5ng/well plasmid DNA.
• the cell media was replaced with serum free medium ( ⁇ Opti-MEM; Invitrogen), and the cells serum starved for either 4 hours (for GPCR studies) or 24 hours (for EGFR studies).
• D-luciferin 50 ⁇ , per well of luciferase substrate, D-luciferin, was then added to a final concentration of 0.2mM D-luciferin, 2mM MgCl 2 , and 2mM ATP, and the cells incubated for 30 minutes at 37°C.
• 0.02mM D-luciferin was used for AT1R and V2R.
• 0.0 ImM D-luciferin was used for the Iuc2-B2AR experiment.
• luminescence was measured on a GLOMAX Multi Plus plate reader set to 0.5s of integration time.
• luminescence was determined by dividing the luminescence value (RLUs) of each sample by the luminescence from the same sample prior to stimulation (SEE FIG. 1 A-D).
• the CRC of epidermal growth factor receptor (EGFR) endocytosis upon treatment with varying concentrations of epidermal growth factor (EGF) was determined from the luminescence of each sample (agonist concentration) at 2 hours normalized to the luminescence value of the same sample prior to stimulation (SEE FIG. 2B).
• the isoproterenol concentration response curves (CRC) of beta2-adrenergic receptor endocytosis in HeLa cells was determined from the luminescence of each sample (agonist concentration) at 20 minutes normalized to the luminescence of the same sample prior to stimulation (SEE FIG. 2C).
• the isoproterenol concentration response curves (CRC) of beta2-adrenergic receptor endocytosis using a D-luciferin derivative PBI3102 was determined from the luminescence of each sample (agonist concentration) at 20 minutes normalized to the luminescence of the same sample prior to stimulation (SEE FIG. 2D).
• the isoproterenol concentration response curves (CRC) of beta2-adrenergic receptor endocytosis using the FFLuc variant, luc2 was determined from the luminescence of each sample (agonist concentration) at 30 minutes normalized to the luminescence of the same sample prior to stimulation (SEE FIG. 2E).
• HEK293 cells were transfected with plasmid DNA encoding OgLuc-receptor fusion using Fugene HD according to the manufacturer's instructions.
• 1 part plasmid DNA was diluted into 1000 parts carrier DNA (pGEM-3Z) to yield 50pg/well plasmid DNA.
• the cell media was replaced with serum free medium ( ⁇ of Opti-MEM), and the cells serum starved for 4 hours.
• OgLuc GPCR fusions also enable measurement of agonist dose-responses (FIG 4). Similar to FFLuc endocytosis, OgLuc-GPCR endocytosis was induced with varying concentrations of agonist (including sphingosine-1 -phosphate (S IP) for EDGl). Normalized luminescence was determined by dividing the luminescence value of each sample by the luminescence from the same sample prior to stimulation. For CRC of various GPCRs, the normalized luminescence at 30 minutes stimulation was used. Data were then normalized to percent activation (lowest values assigned 0% and highest values assigned 100%)(SEE FIG. 4) ⁇
• HEK293 cells were transfected with OgLuc-B2AR or OgLuc-V2R fusion proteins and treated as described above (FIG 5 A-B).
• Known B2AR receptor agonists do not promote endocytosis of OgLuc- V2R fusion proteins (SEE FIG. 5B).
• known V2R agonists do not promote endocytosis of OgLuc- B2AR fusion proteins (SEE FIG. 5A).
• Luciferase-receptor fusions and cell-permeable substrates to monitor inhibition of receptor endocytosis.
• CRCs of isoproterenol-induced endocytosis of OgLuc-B2AR fusions was performed as described in Example 2 except in the presence/absence of ⁇ alprenerol antagonist (added 15 minutes prior to treatment with isoproterenol) (SEE FIG. 6).
• CRCs of isoproterenol-induced endocytosis of FFluc-B2AR fusions was performed as described in Example 1 except in cells overexpressing dominant- negative dynamin (K44A). To co-express dynamin (K44A) and the FFluc-B2AR, the carrier DNA was replaced with plasmid DNA encoding dynamin (K44A) under control of a CMV promoter (SEE FIG. 7).
• receptor recycling e.g., endocytosis and exocytosis.
• receptor recycling could be measured by pulse-chase experiments in which, after inducing reporter endocytosis, the inducing agonist is removed. An increase in luminescence would indicate
• GPCR G-protein coupled receptor
• V2R vasopressin 2 receptor
• B2AR negative control beta2-adrenergic receptor
• HEK293 cells were transfected with plasmid DNA encoding the FFluc-GPCR fusions using Fugene HD (Promega Corp.) according to the manufacturer's instructions.
• 1 part plasmid DNA (by mass) was diluted into 100 parts carrier DNA (pGEM-3Z; Promega Corp.) yielding approximately 0.5ng/well reporter DNA.
• the cell media was replaced with serum free medium ( ⁇ Opti-MEM; Invitrogen), and the cells serum starved for 1 hour. After serum starvation, cells were stimulated with serially diluted arginine vasopressin (A VP) for 30 minutes.
• a VP arginine vasopressin
• Extracellular luciferase was then inhibited via addition of a solution (50 ⁇ 11) of trypsin/EDTA (Gibco) to a final concentration of 0.08% trypsin.
• Cells were incubated 30 minutes at 37°C.
• 50 ⁇ 11 of a solution of D-luciferin (without Mg/ATP) was then added (to a final concentration of 2mM) and cells were incubated an additional 5 hours.
• Dose- dependent increase in raw luminescence signal is observed for V2R fusion proteins, whereas no increase is observed for negative control (B2AR) fusion proteins (See FIG 2F).

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