AU4475400A

AU4475400A - Protein-protein interactions

Info

Publication number: AU4475400A
Application number: AU44754/00A
Authority: AU
Inventors: Paul L. Bartel; Karen Heichman
Original assignee: Myriad Genetics Inc
Current assignee: Myriad Genetics Inc
Priority date: 1999-04-22
Filing date: 2000-04-21
Publication date: 2000-11-10
Also published as: EP1181549A1; CA2371006A1; JP2002542774A; WO2000065340A1

Description

WO 00/65340 PCT/US00/10651 TITLE OF THE INVENTION PROTEIN-PROTEIN INTERACTIONS ) BACKGROUND OF THE INVENTION The present invention relates to the discovery of novel protein-protein interactions that are involved in mammalian physiological pathways, including physiological disorders or diseases. Examples of physiological disorders and diseases include non-insulin dependent diabetes mellitus (NIDDM), neurodegenerative disorders, such as Alzheimer's Disease (AD), and the like. Thus, the 5 present invention is directed to complexes of these proteins and/or their fragments, antibodies to the complexes, diagnosis of physiological generative disorders (including diagnosis of a predisposition to and diagnosis of the existence of the disorder), drug screening for agents which modulate the interaction of proteins described herein, and identification of additional proteins in the pathway common to the proteins described herein. 3 The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated herein by reference, and for convenience, are referenced by author and date in the following text and respectively grouped in the appended List of References. Many processes in biology, including transcription, translation and metabolic or signal 5 transduction pathways, are mediated by non-covalently associated protein complexes. The formation of protein-protein complexes or protein-DNA complexes produce the most efficient chemical machinery. Much of modern biological research is concerned with identifying proteins involved in cellular processes, determining their functions, and how, when and where they interact with other proteins involved in specific pathways. Further, with rapid advances in genome 0 sequencing, there is a need to define protein linkage maps, i.e., detailed inventories of protein interactions that make up functional assemblies of proteins or protein complexes or that make up physiological pathways.

WO 00/65340 PCT/US00/10651 2 Recent advances in human genomics research has led to rapid progress in the identification of novel genes. In applications to biological and pharmaceutical research, there is a need to determine functions of gene products. A first step in defining the function of a novel gene is to determine its interactions with other gene products in appropriate context. That is, since proteins 5 make specific interactions with other proteins or other biopolymers as part of functional assemblies or physiological pathways, an appropriate way to examine function of a gene is to determine its physical relationship with other genes. Several systems exist for identifying protein interactions and hence relationships between genes. There continues to be a need in the art for the discovery of additional protein-protein 0 interactions involved in mammalian physiological pathways. There continues to be a need in the art also to identify the protein-protein interactions that are involved in mammalian physiological disorders and diseases, and to thus identify drug targets. SUMMARY OF THE INVENTION 5 The present invention relates to the discovery of protein-protein interactions that are involved in mammalian physiological pathways, including physiological disorders or diseases, and to the use of this discovery. The identification of the interacting proteins described herein provide new targets for the identification of useful pharmaceuticals, new targets for diagnostic tools in the identification of individuals at risk, sequences for production of transformed cell lines, cellular 0 models and animal models, and new bases for therapeutic intervention in such physiological pathways Thus, one aspect of the present invention is protein complexes. The protein complexes are a complex of (a) two interacting proteins, (b) a first interacting protein and a fragment of a second interacting protein, (c) a fragment of a first interacting protein and a second interacting protein, or Z5 (d) a fragment of a first interacting protein and a fragment of a second interacting protein. The fragments of the interacting proteins include those parts of the proteins, which interact to form a complex. This aspect of the invention includes the detection of protein interactions and the production of proteins by recombinant techniques. The latter embodiment also includes cloned sequences, vectors, transfected or transformed host cells and transgenic animals. 30 A second aspect of the present invention is an antibody that is immunoreactive with the above complex. The antibody may be a polyclonal antibody or a monoclonal antibody. While the antibody is immunoreactive with the complex, it is not immunoreactive with the component parts WO 00/65340 PCT/US00/10651 of the complex. That is, the antibody is not immunoreactive with a first interactive protein, a fragment of a first interacting protein, a second interacting protein or a fragment of a second interacting protein. Such antibodies can be used to detect the presence or absence of the protein complexes. A third aspect of the present invention is a method for diagnosing a predisposition for physiological disorders or diseases in a human or other animal. The diagnosis of such disorders includes a diagnosis of a predisposition to the disorders and a diagnosis for the existence of the disorders. In accordance with this method, the ability of a first interacting protein or fragment thereof to form a complex with a second interacting protein or a fragment thereof is assayed, or the genes encoding interacting proteins are screened for mutations in interacting portions of the protein molecules. The inability of a first interacting protein or fragment thereof to form a complex, or the presence of mutations in a gene within the interacting domain, is indicative of a predisposition to, or existence of a disorder. In accordance with one embodiment of the invention, the ability to form a complex is assayed in a two-hybrid assay. In a first aspect of this embodiment, the ability to form a complex is assayed by a yeast two-hybrid assay. In a second aspect, the ability to form a complex is assayed by a mammalian two-hybrid assay. In a second embodiment, the ability to form a complex is assayed by measuring in vitro a complex formed by combining said first protein and said second protein. In one aspect the proteins are isolated from a human or other animal. In a third embodiment, the ability to form a complex is assayed by measuring the binding of an antibody, which is specific for the complex. In a fourth embodiment, the ability to form a complex is assayed by measuring the binding of an antibody that is specific for the complex with a tissue extract from a human or other animal. In a fifth embodiment, coding sequences of the interacting proteins described herein are screened for mutations. A fourth aspect of the present invention is a method for screening for drug candidates which are capable of modulating the interaction of a first interacting protein and a second interacting protein. In this method, the amount of the complex formed in the presence of a drug is compared with the amount of the complex formed in the absence of the drug. If the amount of complex formed in the presence of the drug is greater than or less than the amount of complex formed in the absence of the drug, the drug is a candidate for modulating the interaction of the first and second interacting proteins. The drug promotes the interaction if the complex formed in the presence of the drug is greater and inhibits (or disrupts) the interaction if the complex formed in the presence of the WO 00/65340 PCT/US00/10651 4 drug is less. The drug may affect the interaction directly, i.e., by modulating the binding of the two proteins, or indirectly, e.g., by modulating the expression of one or both of the proteins. A fifth aspect of the present invention is a model for such physiological pathways, disorders or diseases. The model may be a cellular model or an animal model, as further described herein. In accordance with one embodiment of the invention, an animal model is prepared by creating transgenic or "knock-out" animals. The knock-out may be a total knock-out, i.e., the desired gene is deleted, or a conditional knock-out, i.e., the gene is active until it is knocked out at a determined time. In a second embodiment, a cell line is derived from such animals for use as a model. In a third embodiment, an animal model is prepared in which the biological activity of a protein complex 3 of the present invention has been altered. In one aspect, the biological activity is altered by disrupting the formation of the protein complex, such as by the binding of an antibody or small molecule to one of the proteins which prevents the formation of the protein complex. In a second aspect, the biological activity of a protein complex is altered by disrupting the action of the complex, such as by the binding of an antibody or small molecule to the protein complex which 5 interferes with the action of the protein complex as described herein. In a fourth embodiment, a cell model is prepared by altering the genome of the cells in a cell line. In one aspect, the genome of the cells is modified to produce at least one protein complex described herein. In a second aspect, the genome of the cells is modified to eliminate at least one protein of the protein complexes described herein. O A sixth aspect of the present invention are nucleic acids coding for novel proteins discovered in accordance with the present invention. A seventh aspect of the present invention is a method of screening for drug candidates useful for treating a physiological disorder. In this embodiment, drugs are screened on the basis of the association of a protein with a particular physiological disorder. This association is established in 5 accordance with the present invention by identifying a relationship of the protein with a particular physiological disorder. The drugs are screened by comparing the activity of the protein in the presence and absence of the drug. If a difference in activity is found, then the drug is a drug candidate for the physiological disorder. The activity of the protein can be assayed in vitro or in vivo using conventional techniques, including transgenic animals and cell lines of the present 0 invention.

WO 00/65340 PCT/US00/10651 5 DETAILED DESCRIPTION OF THE INVENTION The present invention is the discovery of novel interactions between proteins described herein. The genes coding for some of these proteins may have been cloned previously, but their potential interaction in a physiological pathway or with a particular protein was unknown. 5 Alternatively, the genes coding for some of these proteins have not been cloned previously and represent novel genes. These proteins are identified using the yeast two-hybrid method and searching a human total brain library, as more fully described below. According to the present invention, new protein-protein interactions have been discovered. The discovery of these interactions has identified several protein complexes for each protein-protein S interaction. The protein complexes for these interactions are set forth below in Tables 1-73, which also identify the new protein-protein interactions of the present invention. TABLE 1 Protein Complexes of Glut4/CARP Interaction 5 Glucose Transporter 4 (Glut4) and Clone C-193 (CARP) A fragment of Glut4 and CARP Glut4 and a fragment of CARP A fragment of Glut4 and a fragment of CARP ) TABLE 2 Protein Complexes of Glutl/DRAL(FHL2) Interaction Glucose Transporter 1 (Glutl) and DRAL(FHL2) A fragment of Glutl and DRAL(FHL2) Glutl and a fragment of DRAL(FHL2) 5 A fragment of Glutl and a fragment of DRAL(FHL2) TABLE 3 Protein Complexes of Glutl/Myosin Heavy Chain Interaction Glucose Transporter 1 (Glutl) and myosin heavy chain 0 A fragment of Glutl and myosin heavy chain Glutl and a fragment of myosin heavy chain A fragment of Glutl and a fragment of myosin heavy chain WO 00/65340 PCT/US00/10651 6 TABLE 4 Protein Complexes of Glutl/HSS Interaction Glucose Transporter 1 (Glutl) and human sperm surface protein (HSS) A fragment of Glutl and HSS 5Glutl and a fragment of HSS A fragment of Glutl and a fragment of HSS TABLE 5 Protein Complexes of OGTase/Mvosin Heavy Chain Interaction O-linked N-acetylglucosaminyltransferase (OGTase) and myosin heavy chain A fragment of OGTase and myosin heavy chain OGTase and a fragment of myosin heavy chain A fragment of OGTase and a fragment of myosin heavy chain 5 TABLE6 Protein Complexes of IRAP/14-3-3 Beta Interaction Insulin-Regulated Membrane-Spanning Aminopeptidase (IRAP) and 14-3-3 beta A fragment of IRAP and 14-3-3 beta IRAP and a fragment of 14-3-3 beta 0 A fragment of IRAP and a fragment of 14-3-3 beta TABLE 7 Protein Complexes of IRAP/HSS Interaction Insulin-Regulated Membrane-Spanning Aminopeptidase (IRAP) and human sperm surface 5 protein (HSS) A fragment of IRAP and HSS IRAP and a fragment of HSS A fragment of IRAP and a fragment of HSS 0 TABLE8 Protein Complexes of PI-3K110/Complement Protein C4 Interaction PI-3 Kinase p110 subunit (PI-3K110) and Complement Protein C4 A fragment of PI-3K110 and Complement Protein C4 PI-3K110 and a fragment of Complement Protein C4 5 A fragment of PI-3K110 and a fragment of Complement Protein C4 WO 00/65340 PCT/US00/10651 7 TABLE 9 Protein Complexes of PI-3K1 10O/Tenascin XB Interaction PI-3 Kinase p 1 10 subunit (PI-3K110) and Tenascin XB A fragment of PI-3K110 and Tenascin XB 5 PI-3K110 and a fragment of Tenascin XB A fragment of PI-3K110 and a fragment of Tenascin XB TABLE 10 Protein Complexes of PI-3K110 O/GAA Interaction ) PI-3 Kinase p 1 10 subunit (PI-3K110) and Alpha Acid Glucosidase (GAA) A fragment of PI-3K110 and GAA PI-3K110 and a fragment of GAA A fragment of PI-3K110 and a fragment of GAA 5 TABLE 11 Protein Complexes of MM-1/C-Napl1 Interaction C-myc Binding Protein (MM-1) and C-Napl A fragment of MM-1 and C-Nap 1 MM-1 and a fragment of C-Napl 0 A fragment of MM-1 and a fragment of C-Nap 1 TABLE 12 Protein Complexes of MM- I/Beta Spectrin Interaction C-myc Binding Protein (MM-1) and Beta Spectrin 5 A fragment of MM-1 and Beta Spectrin MM-1 and a fragment of Beta Spectrin A fragment of MM-1 and a fragment of Beta Spectrin TABLE 13 0 Protein Complexes of MM- 1/KIAA0477 Interaction C-myc Binding Protein (MM-1) and KIAA0477 A fragment of MM-1 and KIAA0477 MM-1 and a fragment of KIAA0477 A fragment of MM-1 and a fragment of KIAA0477 5 WO 00/65340 PCT/US00/10651 8 TABLE 14 Protein Complexes of Dvnamin/CALM Interaction Dynamin and Calthrin Assembly Protein (CALM) A fragment of Dynamin and CALM Dynamin and a fragment of CALM A fragment of Dynamin and a fragment of CALM TABLE 15 Protein Complexes of Dvnamin/Psme3 Interaction ) Dynamin and Proteosome Activator Subunit Psme3 (Psme3) A fragment of Dynamin and Psme3 Dynamin and a fragment of Psme3 A fragment of Dynamin and a fragment of Psme3 5 TABLE 16 Protein Complexes of Naflb/I-TRAF Interaction Nef-Associated Factor 1 beta (Nafl b) and I-TRAF A fragment of Nafl b and I-TRAF Nafl b and a fragment of I-TRAF 0 A fragment of Naflb and a fragment of I-TRAF TABLE 17 Protein Complexes of Aktl/NuMA1 Interaction Akt kinase 1 (Aktl) and NuMAl 5 A fragment of Akt 1 and NuMAl Aktl and a fragment of NuMAl A fragment of Aktl and a fragment of NuMAl TABLE 18 0 Protein Complexes of Akt2/NuMA1 Interaction Akt kinase 2 (Akt2) and NuMA1 A fragment of Akt2 and NuMAl Akt2 and a fragment of NuMAl A fragment of Akt2 and a fragment of NuMAl 5 WO 00/65340 PCT/US00/10651 9 TABLE 19 Protein Complexes of Akt2/BAP31 Interaction Akt kinase 2 (Akt2) and BAP31 A fragment of Akt2 and BAP31 5 Akt2 and a fragment of BAP31 A fragment of Akt2 and a fragment of BAP31 TABLE 20 Protein Complexes of Akt2/Beta Adaptin Interaction 0 Akt kinase 2 (Akt2) and beta adaptin A fragment of Akt2 and beta adaptin Akt2 and a fragment of beta adaptin A fragment of Akt2 and a fragment of beta adaptin 5 TABLE21 Protein Complexes of OGTase/Desmin Interaction O-linked N-acetylglucosaminyltransferase (OGTase) and desmin A fragment of OGTase and desmin OGTase and a fragment of desmin 0 A fragment of OGTase and a fragment of desmin TABLE 22 Protein Complexes of OGTase/Alpha-karvopherin Interaction O-linked N-acetylglucosaminyltransferase (OGTase) and alpha-karyopherin 5 A fragment of OGTase and alpha-karyopherin OGTase and a fragment of alpha-karyopherin A fragment of OGTase and a fragment of alpha-karyopherin TABLE 23 0 Protein Complexes of OGTase/Glutaminyl tRNA Synthetase Interaction O-linked N-acetylglucosaminyltransferase (OGTase) and glutaminyl tRNA synthetase A fragment of OGTase and glutaminyl tRNA synthetase OGTase and a fragment of glutaminyl tRNA synthetase A fragment of OGTase and a fragment of glutaminyl tRNA synthetase 5 WO 00/65340 PCT/US00/10651 10 TABLE 24 Protein Complexes of OGTase/Clone 25100 Interaction O-linked N-acetylglucosaminyltransferase (OGTase) and clone 25100 A fragment of OGTase and clone 25100 OGTase and a fragment of clone 25100 A fragment of OGTase and a fragment of clone 25100 TABLE 25 Protein Complexes of PTP 1 b/VAP-A Interaction 3 PTP 1 b and VAMP-associated protein A (VAP-A) A fragment of PTP 1 b and VAP-A PTP 1 b and a fragment of VAP-A A fragment of PTP 1 b and a fragment of VAP-A 5 TABLE26 Protein Complexes of Rab4/Alpha-catenin-like Protein Interaction Rab4 and alpha-cantein-like protein A fragment of Rab4 and alpha-cantein-like protein Rab4 and a fragment of alpha-cantein-like protein 0 A fragment of Rab4 and a fragment of alpha-cantein-like protein TABLE 27 Protein Complexes of Rab4/Rab2 Interaction Rab4 and Rab2 5 A fragment of Rab4 and Rab2 Rab4 and a fragment of Rab2 A fragment of Rab4 and a fragment of Rab2 TABLE 28 0 Protein Complexes of Glut4/PN7065 Interaction Glucose Transporter 4 (Glut4) and Novel Protein Fragment PN7065 (PN7065) A fragment of Glut4 and PN7065 Glut4 and a fragment of PN7065 A fragment of Glut4 and a fragment of PN7065 5 WO 00/65340 PCT/US00/10651 11 TABLE 29 Protein Complexes of Glut4/PN7386 Interaction Glucose Transporter 4 (Glut4) and Novel Protein Fragment PN7386 (PN7386) A fragment of Glut4 and PN7386 Glut4 and a fragment of PN7386 A fragment of Glut4 and a fragment of PN7386 TABLE 30 Protein Complexes of OGTase/PN6931 Interaction O-linked N-acetylglucosaminyltransferase (OGTase) and Novel Protein Fragment PN6931 (PN6931) A fragment of OGTase and PN6931 OGTase and a fragment of PN6931 A fragment of OGTase and a fragment of PN6931 5 TABLE 31 Protein Complexes ofNaflb/PN7582 Interaction Nef-Associated Factor 1 beta (Naflb) and Novel Protein Fragment PN7582 (PN7582) A fragment of Naflb and PN7582 0 Naflb and a fragment of PN7582 A fragment of Naflb and a fragment of PN7582 TABLE 32 Protein Complexes of OGTase/Talin Interaction 5 O-linked N-acetylglucosaminyltransferase (OGTase) and Talin A fragment of OGTase and Talin OGTase and a fragment of Talin A fragment of OGTase and a fragment of Talin 0 TABLE 33 Protein Complexes of OGTase/MOP2 Interaction O-linked N-acetylglucosaminyltransferase (OGTase) and MOP2 A fragment of OGTase and MOP2 OGTase and a fragment of MOP2 5 A fragment of OGTase and a fragment of MOP2 WO 00/65340 PCT/US00/10651 12 TABLE 34 Protein Complexes of OGTase/Clone 25100 Interaction O-linked N-acetylglucosaminyltransferase (OGTase) and Clone 25100 A fragment of OGTase and Clone 25100 5 OGTase and a fragment of Clone 25100 A fragment of OGTase and a fragment of Clone 25100 TABLE 35 Protein Complexes of OGTase/KIAA0443 Interaction 0 O-linked N-acetylglucosaminyltransferase (OGTase) and KIAA0443 A fragment of OGTase and KIAA0443 OGTase and a fragment of KIAA0443 A fragment of OGTase and a fragment of KIAA0443 5 TABLE36 Protein Complexes of OGTase/EGR1 Interaction O-linked N-acetylglucosaminyltransferase (OGTase) and EGR1 A fragment of OGTase and EGR1 OGTase and a fragment of EGR1 0 A fragment of OGTase and a fragment of EGR1 TABLE 37 Protein Complexes of OGTase/Dvnamin II Interaction O-linked N-acetylglucosaminyltransferase (OGTase) and Dynamin II 5 A fragment of OGTase and Dynamin II OGTase and a fragment of Dynamin II A fragment of OGTase and a fragment of Dynamin II TABLE 38 0 Protein Complexes of OGTase/INT-6 Interaction O-linked N-acetylglucosaminyltransferase (OGTase) and INT-6 A fragment of OGTase and INT-6 OGTase and a fragment of INT-6 A fragment of OGTase and a fragment of INT-6 5 WO 00/65340 PCT/US00/10651 13 TABLE 39 Protein Complexes of OGTase/HSPCO28 Interaction O-linked N-acetylglucosaminyltransferase (OGTase) and HSPCO28 A fragment of OGTase and HSPCO28 OGTase and a fragment of HSPCO28 A fragment of OGTase and a fragment of HSPCO28 TABLE 40 Protein Complexes of OGTase/BAP31 Interaction D O-linked N-acetylglucosaminyltransferase (OGTase) and BAP31 A fragment of OGTase and BAP31 OGTase and a fragment of BAP31 A fragment of OGTase and a fragment of BAP31 5 TABLE 41 Protein Complexes of OGTase/Interferon-Ind Prot Interaction O-linked N-acetylglucosaminyltransferase (OGTase) and Interferon-Ind Protein A fragment of OGTase and Interferon-Ind Protein OGTase and a fragment of Interferon-Ind Protein 0 A fragment of OGTase and a fragment of Interferon-Ind Protein TABLE 42 Protein Complexes of Glut4/Beta-Catenin Interaction Glucose Transporter 4 (Glut4) and Beta-catenin 5 A fragment of Glut4 and Beta-catenin Glut4 and a fragment of Beta-catenin A fragment of Glut4 and a fragment of Beta-catenin TABLE 43 0 Protein Complexes of Glut4/Alpha-SNAP Interaction Glucose Transporter 4 (Glut4) and Alpha-SNAP A fragment of Glut4 and Alpha-SNAP Glut4 and a fragment of Alpha-SNAP A fragment of Glut4 and a fragment of Alpha-SNAP 5 WO 00/65340 PCT/US00/10651 14 TABLE 44 Protein Complexes of Glut4/MAPKKK6 Interaction Glucose Transporter 4 (Glut4) and MAPKKK6 A fragment of Glut4 and MAPKKK6 5 Glut4 and a fragment of MAPKKK6 A fragment of Glut4 and a fragment of MAPKKK6 TABLE 45 Protein Complexes of Glut4/Tropomyosin 3 Interaction 0 Glucose Transporter 4 (Glut4) and Tropomyosin 3 A fragment of Glut4 and Tropomyosin 3 Glut4 and a fragment of Tropomyosin 3 A fragment of Glut4 and a fragment of Tropomyosin 3 5 TABLE46 Protein Complexes of Glutl/DRAL/FHL2 Interaction Glucose Transporter 1 (Glutl) and DRAL/FHL2 A fragment of Glutl1 and DRAL/FHL2 Glutl and a fragment of DRAL/FHL2 0 A fragment of Glutl and a fragment of DRAL/FHL2 TABLE 47 Protein Complexes of Glutl /MYSA Interaction Glucose Transporter 1 (Glutl) and cardiac muscle myosin heavy chain (MYSA) 5 A fragment of Glutl and MYSA Glutl and a fragment of MYSA A fragment of Glutl1 and a fragment of MYSA TABLE 48 .0 Protein Complexes of IRAP/SLAP-2 Interaction Insulin-Regulated Membrane-Spanning Aminopeptidase (IRAP) and SLAP-2 A fragment of IRAP and SLAP-2 IRAP and a fragment of SLAP-2 A fragment of IRAP and a fragment of SLAP-2 35 WO 00/65340 PCT/US00/10651 15 TABLE 49 Protein Complexes of IRAP/SG2NA Interaction Insulin-Regulated Membrane-Spanning Aminopeptidase (IRAP) and SG2NA A fragment of IRAP and SG2NA 5 IRAP and a fragment of SG2NA A fragment of IRAP and a fragment of SG2NA TABLE 50 Protein Complexes of OGTase/14-3-3-epsilon 0 O-linked N-acetylglucosaminyltransferase (OGTase) and 14-3-3-epsilon A fragment of OGTase and 14-3-3-epsilon OGTase and a fragment of 14-3-3-epsilon A fragment of OGTase and a fragment of 14-3-3-epsilon 5 TABLE51 Protein Complexes of PI-3K85/Chromogranin Interaction PI-3 Kinase p85 subunit (PI-3K85) and Chromogranin A fragment of PI-3K85 and Chromogranin PI-3K85 and a fragment of Chromogranin 0 A fragment of PI-3K85 and a fragment of Chromogranin TABLE 52 Protein Complexes of PI-3K85/SLP-76 Interaction PI-3 Kinase p85 subunit (PI-3K85) and SLP-76 5 A fragment of PI-3K85 and SLP-76 PI-3K85 and a fragment of SLP-76 A fragment of PI-3K85 and a fragment of SLP-76 TABLE 53 0 Protein Complexes of PI-3K85/14-3-3-zeta Interaction PI-3 Kinase p85 subunit (PI-3K85) and 14-3-3-zeta A fragment of PI-3K85 and 14-3-3-zeta PI-3K85 and a fragment of 14-3-3-zeta A fragment of PI-3K85 and a fragment of 14-3-3-zeta 5 WO 00/65340 PCT/US00/10651 16 TABLE 54 Protein Complexes of PI-3K85/14-3-3-eta Interaction PI-3 Kinase p85 subunit (PI-3K85) and 14-3-3-eta A fragment of PI-3K85 and 14-3-3-eta 5 PI-3K85 and a fragment of 14-3-3-eta A fragment of PI-3K85 and a fragment of 14-3-3-eta TABLE 55 Protein Complexes of PI-3K85/TACC2 Interaction 0 PI-3 Kinase p85 subunit (PI-3K85) and TACC2 A fragment of PI-3K85 and TACC2 PI-3K85 and a fragment of TACC2 A fragment of PI-3K85 and a fragment of TACC2 5 TABLE56 Protein Complexes of Glut4/MM-1 Interaction Glucose Transporter 4 (Glut4) and C-Myc Binding Protein (MM-1) A fragment of Glut4 and MM-1 Glut4 and a fragment of MM-1 0 A fragment of Glut4 and a fragment of MM-1 TABLE 57 Protein Complexes of Glutl/KIAA0 144 Interaction Glucose Transporter 1 (Glut 1) and KIAA0144 (KIAA) 5 A fragment of Glutl1 and KIAA Glutl and a fragment of KIAA A fragment of Glut 1 and a fragment of KIAA TABLE 58 0 Protein Complexes of Glutl/Dynamin Interaction Glucose Transporter 1 (Glutl) and Dynamin A fragment of Glutl1 and Dynamin Glutl1 and a fragment of Dynamin A fragment of Glutl and a fragment of Dynamin 5 WO 00/65340 PCT/US00/10651 17 TABLE 59 Protein Complexes of Glutl/Clone 25204 Interaction Glucose Transporter 1 (Glutl) and Clone 25204 A fragment of Glutl1 and Clone 25204 5 Glutl and a fragment of Clone 25204 A fragment of Glutl1 and a fragment of Clone 25204 TABLE 60 Protein Complexes of IRAP/VAP-A Interaction 0 Insulin-Regulated Membrane-Spanning Aminopeptidase (IRAP; oxytocinase) and VAMP Associated Protein A (VAP-A) A fragment of IRAP and VAP-A IRAP and a fragment of VAP-A A fragment of IRAP and a fragment of VAP-A 5 TABLE 61 Protein Complexes of OGTase/Nafl a Interaction O-Linked-N-AcetylglucosaminylTransferase (OGTase) and NEF-Associated Factor 1 Alpha (Nafl a) 0 A fragment of OGTase and Nafl a OGTase and a fragment of Nafla A fragment of OGTase and a fragment of Naf la TABLE 62 5 Protein Complexes of OGTase/Alpha-2-Catenin Interaction O-Linked-N-AcetylglucosaminylTransferase (OGTase) and Alpha-2-Catenin A fragment of OGTase and Alpha-2-Catenin OGTase and a fragment of Alpha-2-Catenin A fragment of OGTase and a fragment of Alpha-2-Catenin 0 TABLE 63 Protein Complexes of PI-3K110 /TRIP 15 Interaction PI-3 Kinase p110 subunit (PI-3K110) and TRIP15 A fragment of PI-3K110 and TRIP 15 5 PI-3K110 and a fragment of TRIP15 A fragment of PI-3K110 and a fragment of TRIP 15 WO 00/65340 PCT/US00/10651 18 TABLE 64 Protein Complexes of Glut4/14-3-3 Zeta Interaction Glucose Transporter 4 (Glut4) and 14-3-3 Zeta A fragment of Glut4 and 14-3-3 Zeta 5 Glut4 and a fragment of 14-3-3 Zeta A fragment of Glut4 and a fragment of 14-3-3 Zeta TABLE 65 Protein Complexes of Glut4/KIAA0282 Interaction 0 Glucose Transporter 4 (Glut4) and KIAA0282 (an efp-like protein) A fragment of Glut4 and KIAA0282 Glut4 and a fragment of KIAA0282 A fragment of Glut4 and a fragment of KIAA0282 5 TABLE66 Protein Complexes of Glut4/Tankvrase Interaction Glucose Transporter 4 (Glut4) and Tankyrase A fragment of Glut4 and Tankyrase Glut4 and a fragment of Tankyrase 0 A fragment of Glut4 and a fragment of Tankyrase TABLE 67 Protein Complexes of IRAP/PTPZ Interaction Insulin-Regulated Membrane-Spanning Aminopeptidase (IRAP) and protein tyrosine 5 phosphatase zeta (PTPZ) A fragment of IRAP and PTPZ IRAP and a fragment of PTPZ A fragment of IRAP and a fragment of PTPZ 0 TABLE 68 Protein Complexes of IRAP/3Spectrin Interaction Insulin-Regulated Membrane-Spanning Aminopeptidase (IRAP) and P3-spectrin A fragment of IRAP and P-spectrin IRAP and a fragment of p-spectrin 5 A fragment of IRAP and a fragment of 1-spectrin WO 00/65340 PCT/US00/10651 19 TABLE 69 Protein Complexes of IRAP/PI-3K85 Interaction Insulin-Regulated Membrane-Spanning Aminopeptidase (IRAP) and PI-3 Kinase p85 subunit (PI-3K85) 5 A fragment of IRAP and PI-3K85 IRAP and a fragment of PI-3K85 A fragment of IRAP and a fragment of PI-3K85 TABLE 70 0 Protein Complexes of PP5/HSP89 Interaction Protein Phosphatase 5 (PP5) and Heat Shock Protein 89 (HSP89) A fragment of PP5 and HSP89 PP5 and a fragment of HSP89 A fragment of PP5 and a fragment of HSP89 5 TABLE 71 Protein Complexes of PP5/Tankyrase Interaction Protein Phosphatase 5 (PP5) and Tankyrase A fragment of PP5 and Tankyrase !0 PP5 and a fragment of Tankyrase A fragment of PP5 and a fragment of Tankyrase TABLE 72 Protein Complexes of PI-3K85/Tankvrase Interaction 25 PI-3 Kinase p85 subunit (PI-3K85) and Tankyrase A fragment of PI-3K85 and Tankyrase PI-3K85 and a fragment of Tankyrase A fragment of PI-3K85 and a fragment of Tankyrase 30 TABLE 73 Protein Complexes of PI-3K110/APP Interaction PI-3 Kinase p 1 1 0 subunit (PI-3K110) and Amyloid Precursor Protein (APP) A fragment of PI-3K110 and APP PI-3K110 and a fragment of APP 35 A fragment of PI-3K110 and a fragment of APP WO 00/65340 PCT/US00/10651 20 The proteins newly associated with a particular physiological pathway described herein are used to screen for drug candidates useful for treating a physiological disorder. These proteins are set forth in Table 74. 5 TABLE74 Proteins for Drug Screening and Physiological Disorder PI-3K110 Alzheimer's Disease PI-3K110 Diabetes TRIP 15 Alzheimer's Disease 0 TRIP 15 Diabetes All Others Diabetes The involvement of above interactions in particular pathways is as follows. One of the key questions which must be answered in order to understand and treat non 5 insulin dependent diabetes mellitus (NIDDM) is how glucose uptake is regulated in the cell. It is known that in adipose and muscle tissues there exists an insulin-regulated membrane-spanning glucose transporter called Glut4 that shuttles between the interior of the cell to the plasma membrane by means of vesicle-mediated endocytosis and exocytosis (Garvey et al., 1998; Haruta et al.,1995; Pessin et al., 1999). Fat and muscle cells from diabetic patients appear to be defective 0 in this type of regulated glucose transport (Zierath et al., 1998). The mechanisms by which this transport to the plasma membrane and return back to the interior of the cell are regulated are not well understood. Toward this end, there has been much interest in analyzing the Glut4 protein and the additional factors that participate in glucose uptake in fat and muscle cells. As described below, the yeast two-hybrid assay has been employed as a means of identifying proteins that interact with !5 Glut4 as well as other molecules that have been implicated in the insulin-dependent transport of glucose. Thus, by understanding the regulation of glucose transport in normal cells, medical interventions can be discovered that would serve to increase sugar uptake in the cells of diabetic patients. One approach to understanding how glucose transport is achieved has been to use the Glut4 30 molecule as a molecular bait in the search for other molecules which can interact with it. In this way, it might be possible to find ways to influence the function of Glut4 and perhaps find ways to cause it to go to the plasma membrane and subsequently remove glucose from the outside of the cell and bring it into the interior. Using the yeast two-hybrid assay, we have been able to demonstrate WO 00/65340 PCT/US00/10651 21 that clone C-193 (known as CARP in other systems) can bind to Glut4. CARP is a cytokine inducible gene that reportedly acts as a negative regulator of cardiac-specific genes (Jeyaseelan et al., 1997). This interaction may serve as a tie between heart disease and diabetes. Using the yeast two-hybrid assay, we have also been able to detect the interaction of Glut4 5 with two novel proteins that have been named PN7065 and PN7386. PN7065 bears a striking similarity to a rat salt-induced protein kinase (GenBank accession AB020480). Experiments in rats have shown that this salt-induced kinase may play an important role in the regulation of adrenocortical functions in response to high plasma salt and ACTH stimulation (Wang et al., 1999). It is possible that Glut4 may act as a substrate for the kinase. PN7386 is identical to a human 0 chromosome 20 clone called 850H21 (GenBank accession ALO31680) that is uncharacterized in the literature. It is possible that the protein product of this clone may participate in protein trafficking or in the signal transduction mechanism that regulates this process. Using the yeast two-hybrid assay, we have been able to detect four more proteins, beta catenin, alpha-SNAP, tropomyosin 3 and MAPKKK6, which can bind to Glut4. Beta-catenin is a 5 protein containing so-called Armadillo repeats that is involved in two important cellular processes: signal transduction via the Wingless pathway and cell adhesion (Ben-Ze'ev et al., 1998). This interaction between Glut4 and beta-catenin may shed light on the regulation of insulin-responsive glucose transport since it links the transporter to an important signaling pathway. The alpha-SNAP is an important mediator in the cellular process of intracellular transport (St-Denis, et al., 1998). .0 The finding that alpha-SNAP and Glut4 interact provides a link between glucose transportation and the machinery required to perform movement of the glucose transporter between the outside and the interior of the cell. Glut4 has been demonstrated to interact with tropomyosin 3, a protein involved in muscle contraction (Squire et al., 1998). This interaction may represent a link between glucose uptake and muscle function. Finally, the Glut4 glucose transporter has been shown to interact with .5 a putative protein kinase, MAPKKK6. This enzyme has not been well-characterized, but it was identified by virtue of its ability to bind to another protein kinase (Wang et al., 1998). Once again, this may provide a clue to the mechanism of regulation of glucose transport since Glut4 can interact with another potential signal tranduction mediator. Using the yeast two-hybrid assay, we have been able to detect three proteins which can bind 30 to Glut4, 14-3-3 zeta, the efp-like protein (KIAA0282) and tankyrase. The 14-3-3 zeta is a signal transduction protein which has been shown to interact specifically with phospherine residues (Thorson et al., 1998). 14-3-3 zeta is part of a pathway that links the insulin receptor molecule at WO 00/65340 PCT/US00/10651 22 the cell surface to the Glut4 protein located either in the interior of the cell or also at the cell surface. Interestingly, the same small region of Glut4 that interacts with 14-3-3 zeta has been shown to be phosphorylated on a critical serine residue by the kinase Akt-2 that has also been implicated in glucose uptake (Kupriyanova et al., 1999). The efp-like protein is a putative transcription factor 5 (Orimo et al., 1995). Its function is not well described but the Glut4 protein could influence the transcriptional activation of various genes, some of which might be involved in cellular metabolism. Tankyrase is a known telomere-asscoiated protein (Smith et al., 1998). Using the yeast two-hybrid assay, we have been able to detect an additional protein, which can bind to Glut4. This protein is called MM-1 and was identified by virtue of its ability to interact 0 with the proto-oncogene c-myc (Mori et al., 1998). Other than this original characterization, there is not much else known about MM-1 but on the basis of the interaction found here, MM-1 may play critical roles in both cancer and diabetes. Other than this original characterization, there was not much else known about MM-1, however because of its association with Glut4 and its tie to Diabetes, we have used MM-1 in two 5 hybrid assays. Using the yeast two-hybrid assay, we have identified the large centrosomal protein C-Napl as an interactor ofMM-1. C-Nap1 was originally identified as a protein that could interact with the Nek2 cell cycle-regulated protein kinase (Fry et al., 1998). The finding that MM-1 can interact with C-Napl serves to tie Glut4 and glucose transport in general to the control of the cell cycle. The second protein shown to interact with MM-1 is beta spectrin. Spectrins give flexibility 0 to the cell and also act as a scaffold for other cellular proteins (Grum et al., 1999). Interestingly, we have linked beta spectrin to glucose transport and Diabetes in a previous finding where it was shown that beta spectrin could interact with the vesicle-associated protein IRAP. The finding that MM-1 can bind to beta spectrin further strengthens the argument that beta spectrin plays a role in glucose transport. MM-1 was shown to bind to a third protein, KIAA0477, which has no known function. 5 KIAA0477 was originally isolated from brain but its tissue distribution is not known. The finding that KIAA0477 interacts with MM-1 suggests that KIAA0477 plays a role in glucose transport or in some cellular function associated with vesicular transport. Another approach to understanding the mechanism of glucose transport has been to use the Glutl molecule (Hresko et al., 1994), a gene highly related to Glut4 and possessing similar 0 biological function, as a molecular bait in the search for other molecules that can interact with it. Using the yeast two-hybrid assay, we have identified three more proteins which can bind to Glut l: DRAL, HSS and myosin heavy chain. DRAL is a LIM domain-containing protein that is also WO 00/65340 PCT/US00/10651 23 known as FHL-2 or SLIM3. DRAL was identified as a protein that was expressed in normal muscle tissue culture cells but was down-regulated in cancerous rhabdomyosarcoma cells (Genini et al., 1997). It is possible that DRAL plays a critical role in the terminal differentiation of muscle cells such as cardiac muscle, and that its misregulation could result in an undifferetiated cancerous phenotype. Glutl was also shown to interact with HSS or human sperm surface protein. HSS is a testis-specific protein that has no known function (Shankar et al., 1998). It does contain a putative transmembrane domain and a leucine-zipper dimerization domain. Since it interacts with Glutl1 and it is presumably membrane-bound, HSS could potentially act with Glutl or Glut4 to affect glucose transport in the testis. Glutl1 has also been demonstrated to interact with a form of the myosin heavy S chain. Myosin heavy chain plays a key role in muscle structure and contraction (Eddinger et al., 1998). The interaction between Glutl1 and myosin suggests that glucose uptake and muscle function may be interrelated via the association of these two proteins. Using the yeast two-hybrid assay, we identified dynamin as an interactor of Glutl. Dynamin has been implicated in the movement of glucose transporters via vesicular trafficking and likely 5 plays a critical role in endocytosis (or movement from the cell surface to the interior of the cell) (Kao et al., 1998). Because of dynamin's link to Diabetes and glucose transport, we used it in two hybrid assays and found two proteins that could interact with it. The first protein is called CALM, and it is a clathrin assembly protein similar to the AP-3 family of adaptor proteins. CALM was originally found in a lymphoid myeloid leukemia cell line containing a chromosome translocation 0 resulting in the fusion of the AF10 gene with CALM (Dreyling et al., 1996). Clathrin and its associated proteins have a long history of involvement in the transport of vesicles from the cell surface to the interior of the cell. The association of dynamin and CALM further supports this role and ties CALM to glucose transport. Dynamin also binds to a proteosome activator subunit termed Psme3. The human Psme3 gene maps to the region of the BRCA1 gene, and its function was 5 deduced by its similarity to the mouse gene that is also referred to as the Ki antigen (Kohda et al., 1998). Since the proteosome is required for the post-translational processing and the specific degradation of certain proteins, the finding that Psme3 can bind to dynamin implies that this type of protease activity may play a key role in glucose transport. Using the yeast two-hybrid assay, we have identified two more proteins which can bind to 0 Glutl, DRAL/FHL2 and cardiac muscle myosin heavy chain. The first protein, DRAL/FHL2, is a protein shown to be down-regulated in rhabdomyosarcoma (Genini et al., 1997). It is entirely composed of LIM domains, polypeptide motifs that form double zinc fingers and may function by WO 00/65340 PCT/US00/10651 24 facilitating binding to nucleic acid or other proteins. The same region of Glutl has been shown to bind to a cardiac muscle myosin heavy chain (MYSA)(Metzger et al., 1999). The significance of this interaction is unknown with regard to glucose transport, however myosin is known to function in muscle contraction and cell structure. Using the yeast two-hybrid assay, we have identified three proteins which can bind to Glutl, dynamin, and two proteins of unknown function, KIAA0144 and clone 25204. Dynamin has been implicated in the movement of glucose transporters via vesicular trafficking and likely plays a critical role in endocytosis (or movement from the cell surface to the interior of the cell) (Kao et al., 1998). Although the KIAA0144 gene is uncharacterized to date, the region of it that contacts Glut 1 S is highly enriched for serine, threonine and proline residues, possibly providing a clue to its function. Other proteins with similar "STP" domains include the extracellular portions of cell surface receptors. Finally, a potential translation product of clone 25204 bears a striking resemblance to a previously identified mouse gene called SEZ-6. This gene was found by virtue of its increased transcript levels in brain tissue following exposure to a seizure producing drug 5 (Shimizu-Nishikawa et al., 1995). The insulin-regulated membranse-spanning aminopeptidase or IRAP (also known as vp 165, gp160 and oxytocinase) co-localizes with the Glut4 transporter in specified endocytic vesicles (Keller et al., 1995; Malide et al., 1997). Since expression of the N-terminal fragment of IRAP has been shown to result in the translocation of Glut4 to the plasma membrane, IRAP is thought to play 0 a key role in glucose transport (Waters et al., 1997). Using the two-hybrid system, we have detected the interaction of IRAP with two proteins, 14-3-3 beta and HSS. The 14-3-3 family of proteins are critical signal transduction proteins that bind to phosphoserine residues (Jin et al., 1996). The interaction of IRAP with 14-3-3 beta strongly suggests that the function of IRAP could be regulated by phosphorylation and by the subsequent binding by 14-3-3 family members. Since IRAP and the 5 Glut4 glucose transporter co-localize in the same intracellular vesicles, it is possible that Glut4 may participate in signal transduction mechanisms mediated by the 14-3-3 proteins such as 14-3-3 beta. IRAP has also been shown to bind to the HSS protein described above. Like Glutl and Glut4, IRAP is membrane-bound and could potentially bind to HSS in the membrane. This finding points to HSS as playing a role in glucose transport or in other important functions performed in intracellular 0 vesicles. Using the two-hybrid system, we have detected the interaction of IRAP with two more proteins. The N-terminal portion of IRAP has been shown to interact with SLAP-2. The rabbit WO 00/65340 PCT/US00/10651 25 homolog of SLAP-2 has been demonstrated to localize to the sarcolemma or the membrane of muscle cells although its function has not been elucidated (Wigle et al., 1997). SLAP-2 may play a role in vesicular transport or may at least participate in it since it has been shown to be membrane associated and localizes to both the cell membrane as well as to intracellular stores in the 5 endoplasmic reticulum. The C-terminal extracellular portion of IRAP has been demonstrated to interact with SG2NA. SG2NA is a cell cycle nuclear autoantigen that contains so-called WD-40 repeats that are present in a variety of signal transduction proteins (Muro et al., 1995). Once again, the significance of this interaction is unclear however it is possible that SG2NA binding to IRAP is part of a more complex regulatory mechanism. D Using the two-hybrid system, we have detected the interaction of IRAP with one more protein. The N-terminal portion of IRAP has been shown to interact VAMP-associated protein A (VAP-A or VAP-33). This protein has been implicated in intracellular transport (specifically exocytosis or movement to the cell surface) in A. californica and likely plays a similar role in humans (Skehel et al., 1995; Weir et al., 1998). 5 Using the two-hybrid system, we have detected the interaction of IRAP with three proteins. The N-terminal portion of IRAP has been shown to interact with another signal transduction protein, the zeta polypeptide of protein tyrosine phosphatase. This protein is not well characterized but could play a role in regulating glucose transport by dephosphorylating critical proteins that cause or prevent glucose transport (Nishiwaki et al., 1998). The C-terminal portion of IRAP has been 0 shown to interact with non-erythrocytic beta-spectrin. This protein is thought to be involve in secretion and could play a role in the movement of Glut 4 vesicles through IRAP (Hu et al., 1992). The C-terminus of IRAP has also been shown in the two-hybrid assay to interact with the p85 regulatory subunit of phosphatidylinositol-3 (PI-3) kinase. This protein is a central player in cellular signal transduction and participates in transmitting signals from the outside of the cell into the 5 interior. Interestingly, one of the functions of PI-3 kinase p85 involves the insulin receptor (Martin et al., 1996). Further, it is well known that the movement of Glut 4 between the plasma membrane and the interior of the cell depends on the action of the PI-3 kinase signal transduction pathway since inhibitors of this kinase prevent the cycling of Glut4. We also discovered that PI-3 kinase p85 interacts with tankyrase. Two-hybrid interactions have been detected using the p 10 catalytic 0 subunit of PI-3 kinase, and these include interactions with the p85 and p55 subunits of the same enzyme complex as well as non-subunit interactions.

WO 00/65340 PCT/US00/10651 26 Protein phosphatase 5 (PP5) is a TPR domain containing protein that seems to be part of larger multiprotein complexes that possess several cellular functions (Silverstein et al., 1997). Our two-hybrid studies have confirmed the biochemical interaction between protein phosphatase 5 and Hsp90, and the association between these proteins has been previously demonstrated using 5 biochemical methods. PP5 has also been demonstrated using the two-hybrid assay herein to interact with another related heat shock protein, Hsp89, and also with tankyrase. Phosphatidyl inositol-3 kinase is a very important signal transduction protein and likely plays a critical role in Glut4-mediated glucose uptake (Shankar et al.. 1998). This protein participates in transmitting signals from the outside of the cell into the interior. It is composed of 0 two subunits, the p85 regulatory subunit and the p110 catalytic subunit, and functions by facilitating the transmission of signals from the outside of the cell into the interior. PI-3 kinase has been implicated in insulin-regulation and glucose uptake since one of its functions involves the insulin receptor (Martin et al., 1996). Further, the movement of Glut4 between the plasma membrane and the interior of the cell depends on the action of the PI-3 kinase signal transduction pathway since 5 inhibitors of this kinase prevent the cycling of Glut4. The p85 regulatory subunit of PI-3 kinase has been shown to interact with several proteins in the two-hybrid assay. Here we report the identification of five more proteins that can interact with p85. SLP-76 is a tyrosine phosphoprotein that participates in T cell signaling (Clements et al., 1998; Jackman et al., 1995). It is thought that SLP-76 acts as a so-called adaptor protein since 0 it plays a role in intermediate steps of signal transduction. This is achieved by bridging factors that act at the plasma membrane with other molecules that perform functions within the interior of the cell. Our results indicate that SLP-76 may play a critical role in the PI-3 kinase signal transduction pathway by virtue of its ability to bind the p85 regulatory subunit. The p85 subunit of PI-3 kinase has also been demonstrated to bind to two more important signal transduction proteins: 14-3-3 zeta 5 and 14-3-3 eta. These proteins bind specifically to phosphoserine residues in a number of proteins (Oghira et al., 1997; Thorson et al., 1998; Yaffe et al., 1997). Interestingly, our studies have shown that the Glut4 glucose transporter can also interact with 14-3-3 zeta. Thus, PI-3 kinase can also be connected to glucose uptake mechanisms by its interaction with the 14-3-3 signal transduction proteins. PI-3 kinase p85 was shown to interact with chromogranin C, a neuroendocrine secretory 0 granule protein in the granin family (Ozawa et al., 1995). Members of the granin family localize to specialized secretory vesicles and are thought to serve an important function in protein sorting and secretion (Leitner et al., 1999). Finally, TACC2 has been shown to interact with PI-3 kinase WO 00/65340 PCT/US00/10651 27 p85 in the yeast two-hybrid assay. TACC2 is a member of a family of "transforming coiled coil" proteins that have been implicated in cellular growth control and cancer (Still et al., 1999). Although the function of TACC2 remains unknown, its interaction with p85 demonstrates that it may also be a part of an important signal transduction pathway. 5 The p 1 10 subunit of this protein has been shown to interact with complement protein C4, tenascin XB and alpha acid glucosidase (GAA). The complement C4 protein plays a key role in acitvating the classical complement pathway, and it is involved in evoking histamine release from basophils and mast cells. Naturally occurring deficiencies of C4 have been correlated with a number of immune-related human diseases such as Systemic Lupus Erythmatosus, kidney disease, hepatitis, 0 dementia and the propensity for recurrent infectiions (Mascart-Lemone et al., 1983; Vergani et al., 1985; Waters et al., 1997; Nerl et al., 1984; Lhotta et al., 1990). The finding that C4 interacts with the p1 10 subunit of PI-3 kinase suggests that the function of C4 is somehow regulated by this signal transduction, perhaps in response to some immunologic stimulus. The p1 10 catalytic subunit of PI 3 kinase has been demonstrated to interact with tenascin XB. Tenascin XB is an extracellular 5 structural protein. Deficiency oftenascin XB is linked to the connective tissue disorder Ehler-Danlos syndrome (Burch et al., 1997). This finding that p110 can bind to tenascin XB suggest that the function of tenascin XB could be modified or regulated by the PI-3 kinase signal trasduction pathway. PI-3 kinase p 110 has also been shown to bind to GAA or alph acid glucosidase. GAA is a lysosomal enzyme that catalyzes the release of glucose from glycosylated substrates, and defects 0 in GAA result in a glycogen storage disease (Raben et al., 1995). The finding that PI-3 kinase can bind to GAA suggests that GAA activity may be influenced by the PI-3 kinase signaling mechanism. Our previous two-hybrid results have linked the PI-3 kinase to human diseases such as Diabetes and Alzheimer's disease. These new findings therefore link complement protein C4, tenascin XB and alpha acid glucosidase to these diseases as well as to the other diseases already 5 described. The p110 subunit of this protein has been shown to interact with the thyroid hormone interacting protein TRIP 15 in the two-hybrid assay. TRIP 15 is part of a larger multiprotein complex termed the signalsome that is likely to be involved in cell signaling (Seeger et al., 1998). Our two hybrid results have linked the PI-3 kinase to human diseases such as Diabetes and Alzheimer's 0 disease. We have detected the interaction of PI-3 kinase p 110 subunit with the P3-amyloid precursor protein (APP). In brief, there is now growing evidence that APP metabolism and AP3 generation are WO 00/65340 PCT/US00/10651 28 central events to AD pathogenesis. For a more extensive review of APP and AD pathogenesis, see U.S. patent application Serial No. 09/466,139, filed 21 December 1999 and international patent application No. PCT/US99/30396, filed 21 December 1999, each incorporated herein by reference. Although several candidates (cathepsin G, cathepsin D, chymotrypsin, and others) have been 5 suggested, the P3-secretase enzyme has not yet been identified. Even less is known about c- and 7y secretases. The biochemical link between the presenilins and APP processing has not been established. The proteins that mediate the neurotrophic and neuroprotective effects of sAPP are unknown. This last point is of utmost importance because an alteration of APP metabolism could result in both the generation of a toxic product (AP3) and the impairment of sAPP trophic activity 0 (Saitoh et al.1994; Roch et al.1993; Saitoh and Roch, 1995). In this respect, it is interesting that one APP mutation associated with Alzheimer's results in a defective neurite extension activity of sAPP (Li et al., 1997). Moreover, the balance of phosphorylation cascades is deeply altered in Alzheimer brains (Saitoh and Roch, 1995; Jin and Saitoh, 1995; Mook-Jung and Saitoh, 1997; Saitoh et al., 1991; Shapiro et al., 1991). Since APP is thought to play a major role in the pathology of 5 Alzheimer's disease, the elucidation of a tie between PI-3 kinase and APP provides a new target for discovering the treatment for this neurological disorder (Russo et al., 1998). O-linked N-acetylglucosaminyltransferase or OGTase is an enzyme implicated in intracellular signal transduction (Kreppel et al., 1997). It has been speculated that OGTase may play a key role in glucose uptake and may therefore participate in the Diabetes related pathways 10 (Cooksey et al., 1999). OGTase has been shown to also interact with myosin heavy chain. The binding of myosin heavy chain to OGTase suggests that the function of myosin in muscle structure or function could be influenced by OGTase in its signal transductive capacity. This could potentially affect many cellular processes in the muscle cell, including glucose transport. OGTase has been shown to bind to a novel protein termed PN6931. PN6931 is very similar to the mouse !5 kinesin light chain gene (GenBank accession AF055666). Kinesin is a molecular motor involved in cellular transport and chromosome movement (Kirchner et al., 1999). Perhaps by post translationally modifying this novel kinesin-like protein, OGTase can influence its function. Amino acids 250 to 450 of OGTase has been shown to interact with a member of the 14-3-3 protein family, 14-3-3 epsilon. The 14-3-3 proteins function in intracellular signal transduction 30 pathways by specifically binding to phosphoserine residues on other critical signaling molecules (Ogihara et al., 1997; Yaffe et al., 1997). The interaction between OGTase and 14-3-3 epsilon may WO 00/65340 PCT/US00/10651 29 serve to link two different signal transduction pathways, one which involves phosphorylation and a second which involves another type of protein modification. Amino acids 250 to 450 of OGTase has been shown to interact with two proteins, alpha-2 catenin and Nafl a. Alpha-catenin is a protein related to vinculins that functions in cell-cell contact 5 by binding to cadherins (Rudiger et al., 1998). The same region of OGTase can interact with Nafl b, a protein identified by virtue of its ability to bind the Nef gene product of HIV in the two-hybrid assay (Fukushi et al., 1999). Over-expression of Nafla was observed to cause an increase in cell surface expression of the CD4 antigen, therefore this protein may also function in intracellular trafficking and could potentially participate in a number of diseases related to this general process 0 such as Diabetes and Alzheimer's. Using the yeast two-hybrid assay, we identified Nafl b as an interactor of OGTase. Nafl b is a protein which was identified by virtue of its ability to bind the Nef gene product of HIV in the two-hybrid assay (Fukushi et al., 1999). Over-expression of Naflb was observed to cause an increase in cell surface expression of the CD4 antigen, therefore this protein may also function in 5 intracellular trafficking and could potentially participate in a number of diseases related to this general process such as Diabetes and Alzheimer's. In a two-hybrid search using Naflb as a bait, the TRAF-interacting protein I-TRAF was found to be an interactor. I-TRAF appears to act as a regulator of the TRAF signal transduction pathway that transmits signals from TNF (tumor necrosis factor) receptor family members (Rothe et al., 1996). The observation that Naflb interacts with I 0 TRAF serves to link an extracellular stimulated signal transduction mechanism with the OGTase pathway involved in glucose transport. A two-hybrid search using Naflb as a bait has also identified a novel protein fragment called PN7582. This small protein fragment does not appear to have any strong similarity to known proteins therefore its function is not readily apparent. It is very possible that it may participate in protein trafficking or signal transduction by its association with 5 Naflb. In a two-hybrid search using OGTase as a bait, four new interactors were found. The first interactor, desmin, is a cytoplasmic intermediate filament protein found in muscle (Capetanaki et al., 1998). It functions in striated muscle by connecting myofibrils with themselves and with the plasma membrane. Desmin is broken down into three functional domains, the head, rod and tail, 0 and OGTase can bind to the rod structure. The second protein shown to interact with OGTase is called alpha-karyopherin. Alpha-karyopherin (also known as importin and SRP1) is a ubiquitously expressed protein that plays a role in trafficking nuclear localization signal-containing proteins into WO 00/65340 PCT/US00/10651 30 the nucleus through the nuclear pore (Moroianu, 1997). The third protein that OGTase has been shown to bind is glutanimyl-tRNA synthetase. The aminoacyl-tRNA synthetases not only play a key role in protein synthesis, but recent studies have shown that they also impact a number of other cellular processes such as tRNA processing, RNA splicing, RNA trafficking, apoptosis, and transcriptional and translational regulation (Martinis et al., 1999). The fourth protein shown to bind to OGTase in the yeast two-hybrid assay is called clone 25100 and it has no known function. The gene for clone 25100 was isolated from human infant brain and appears to encode a small protein with no structural characteristics that shed light on its function. All four of these OGTase interacting proteins may act as substrates for OGTase or may affect its function in some way. The S finding that they interact with OGTase suggests that they may play a role in glucose transport or in the pathogenesis of Diabetes. Our yeast two-hybrid studies have demonstrated that OGTase can interact with a variety of proteins that can fall into a number of functional categories. Two proteins that have been implicated in vesicular transport bind to OGTase. The first is called BAP31, and it likely plays a critical role 5 in sorting and transporting membrane proteins between intracellular compartments (Annaert et al., 1997). The second protein is called dynamin II, and it is implicated in the movement of transport vesicles from the plasma membrane to sites within the interior of the cell (Sontag et al., 1994). OGTase has been demonstrated to interact with two proteins that function in transcription, as well. The first is called EGR1 for early growth response protein 1, and it has been shown to be highly D induced in cells following mitogenic stimulation (Sukhatme et al., 1988). It is a zinc-finger containing protein, and following its own stimulation, goes on to activate the transcription of genes involved in mitogenesis and differentiation. Additionally, OGTase can interact with MOP2, a basic helix-loop-helix containing transcription factor that is involved in the induction of oxygen regulated genes (Hogenesch et al., 1997). OGTase binds to a structural protein called talin. Talin is a 5 cytoplasmic protein that serves to link integrins with the actin cytoskeleton (Calderwood etal., 1999). Finally, OGTase has been shown in the yeast two-hybrid assay to bind to five proteins of unknown or poorly characterized function. OGTase binds to Int-6, a protein that has also been demonstrated to bind to the HTLV-I Tax transactivator and it is a component of promyelocytic leukemia nuclear bodies (Desbois et al., 1996). OGTase binds to interferon-induced protein 54, an 0 uncharacterized protein that shows a large increase in its transcript following treatment with alpha and beta-interferons (Levy et al., 1986). Lastly, HSPCO28, KIAA0443, and clone 25100 interact with OGTase.

WO 00/65340 PCT/US00/10651 31 Akt kinase is a serine/threonine protein kinase that has been implicated in insulin-regulated glucose transport and the development of non-insulin dependent diabetes mellitus (Krook et al., 1998). Because of this link, Akt kinase has been used in two-hybrid assays to determine what proteins interact with it either because they are substrates of Akt kinase or because they are 5 regulators of the kinase. Two closely related Akt proteins were used: Aktl and its closely related family-member Akt2. Aktl and Akt2 were both shown to interact with the nuclear mitotic apparatus protein NuMA1. NuMA1 displays a distinct pattern of immunofluorescent staining that varies throughout the cell cycle. It is nuclear throughout interphase but re-localizes to the spindle apparatus during mitosis (Lydersen et al., 1980). Phosphorylation is thought to play a critical role 0) in NuMA function. It appears to become phosphorylated just prior to mitosis and becomes dephosphorylated after mitosis has occurred in the G1 phase of the cell cycle mitosis (Sparks et al., 1995). Aktl and Akt2 may be capable of phosphorylating NuMA, especially since the region of NuMA that interacts with Akt2 (amino acids 98 to 365) contains 23 serines and 9 threonines. Akt2 was also shown to interact with two proteins involved in vesicular transport. The first protein, 5 BAP31, likely plays a role in the movement of membrane-bound proteins from the Golgi appartus to the plasma membrane (Annaert et al., 1997). Since BAP31 was also shown to interact with OGTase in our previous experiments, two signal transduction proteins implicated in glucose transport have been tied to BAP31. Akt2 was also shown to bind to another vesicular transport protein termed beta-adaptin or AP2-beta. Beta-adaptin is a part of the AP2 coat assembly complex 0 that links clathrin and to transmembrane receptors resident in coated vesicles (Pearse, 1989). This finding suggests a role for Akt2 in the regulation of endocytosis, while the finding that Akt2 binds to BAP31 provides for a tie between Akt2 and exocytosis. All of the proteins that can interact with the Akt kinase may play a role in glucose transport by virtue of their association with Akt. PTPlb is a protein tyrosine phosphatase that plays a critical role in signal transduction. It 5 has been implicated as a negative regulator of insulin-responsive glucose transport (Chen et al., 1997), and therefore it has been used in yeast two-hybrid assays in an attempt to find more proteins involved in this function, perhaps by acting as substrates. PTPlb was shown to interact with VAMP-associated protein A (VAP-A or VAP-33). This protein has been implicated in intracellular transport (specifically exocytosis or movement to the cell surface) in,4 A. californica and likely plays 0 a similar role in humans (Skehel et al., 1995; weir et al., 1998). The interaction between PTPlb and VAP-A serves as a potential tie between PTPlb and IRAP since IRAP was also shown to bind to VAP-A in two-hybrid experiments. IRAP, insulin-regulated membrane-spanning aminopeptidase WO 00/65340 PCT/US00/10651 32 (also known as vp165, gpl60 and oxytocinase) co-localizes with the Glut4 transporter in specified endocytic vesicles (Keller et al., 1995; Malide et al., 1997). Since expression of the N-terminal fragment of IRAP has been shown to result in the translocation of Glut4 to the plasma membrane, IRAP is thought to play a key role in glucose transport (Waters et al., 1997). Thus, the result that PTPlb interacts with VAP-A serves to strengthen the tie between PTPlb and VAP-A to glucose transport and Diabetes. The small GTP-binding protein Rab4 is another signal transduction factor that has been implicated in the regulation of insulin-stimulated glucose uptake (Vollenweider et al., 1997; Cormont et al., 1996). Rab4, therefore, has also been used in the yeast two-hybrid assay to find ) additional proteins involved in glucose transport and in Diabetes. Two proteins have been shown to bind to Rab4, an alpha-catenin-like protein (sometimes called alpha-catulin) and another small GTP-binding protein called Rab2. The alpha-catenin-like protein resembles alpha-catenin and vinculin however its function has not yet been well-characterized (Janssens et al., 1999). Alpha catenin itself is a protein related to vinculins that functions in cell-cell contact by binding to 5 cadherins (Rudinger et al., 1998). Interestingly, alpha-catenin was found in our previous studies to bind to OGTase and, as a consequence, has been implicated in glucose transport. The second protein shown to bind to Rab4 is Rab2. These two small GTP-binding proteins are highly related with a 50% amino acid identity. Unlike Rab4, Rab2 has not been thought to play a role in insulin stimulated glucose transport (Uphues et al., 1994) although it does play an important role in ) vesicular transport in the cell (Tisdale et al., 1998). The finding that Rab4 and Rab2 interact suggests that they may be capable of influencing each others cellular functions, thus Rab2 could potentially affect Rab4's role in glucose transport. The proteins disclosed in the present invention were found to interact with their corresponding proteins in the yeast two-hybrid system. Because of the involvement of the 5 corresponding proteins in the physiological pathways disclosed herein, the proteins disclosed herein also participate in the same physiological pathways. Therefore, the present invention provides a list of uses of these proteins and DNA encoding these proteins for the development of diagnostic and therapeutic tools useful in the physiological pathways. This list includes, but is not limited to, the following examples.

)

WO 00/65340 PCT/US00/10651 3.3 Two-Hybrid System The principles and methods of the yeast two-hybrid system have been described in detail elsewhere (e.g., Bartel and Fields, 1997; Bartel et al., 1993; Fields and Song, 1989; Chevray and Nathans, 1992). The following is a description of the use of this system to identify proteins that 5 interact with a protein of interest. The target protein is expressed in yeast as a fusion to the DNA-binding domain of the yeast Gal4p. DNA encoding the target protein or a fragment of this protein is amplified from cDNA by PCR or prepared from an available clone. The resulting DNA fragment is cloned by ligation or recombination into a DNA-binding domain vector (e.g., pGBT9, pGBT.C, pAS2-1) such that an in 10 frame fusion between the Gal4p and target protein sequences is created. The target gene construct is introduced, by transformation, into a haploid yeast strain. A library of activation domain fusions (i.e., adult brain cDNA cloned into an activation domain vector) is introduced by transformation into a haploid yeast strain of the opposite mating type. The yeast strain that carries the activation domain constructs contains one or more Gal4p-responsive reporter 15 gene(s), whose expression can be monitored. Examples of some yeast reporter strains include Y190, PJ69, and CBY14a. An aliquot of yeast carrying the target gene construct is combined with an aliquot of yeast carrying the activation domain library. The two yeast strains mate to form diploid yeast and are plated on media that selects for expression of one or more Gal4p-responsive reporter genes. Colonies that arise after incubation are selected for further characterization. 20 The activation domain plasmid is isolated from each colony obtained in the two-hybrid search. The sequence of the insert in this construct is obtained by the dideoxy nucleotide chain termination method. Sequence information is used to identify the gene/protein encoded by the activation domain insert via analysis of the public nucleotide and protein databases. Interaction of the activation domain fusion with the target protein is confirmed by testing for the specificity of the 25 interaction. The activation domain construct is co-transformed into a yeast reporter strain with either the original target protein construct or a variety of other DNA-binding domain constructs. Expression of the reporter genes in the presence of the target protein but not with other test proteins indicates that the interaction is genuine. In addition to the yeast two-hybrid system, other genetic methodologies are available for the 30 discovery or detection of protein-protein interactions. For example, a mammalian two-hybrid system is available commercially (Clontech, Inc.) that operates on the same principle as the yeast two hybrid system. Instead of transforming a yeast reporter strain, plasmids encoding DNA-binding and WO 00/65340 PCT/US00/10651 34 activation domain fusions are transfected along with an appropriate reporter gene (e.g., lacZ) into a mammalian tissue culture cell line. Because transcription factors such as the Saccharomyces cerevisiae Gal4p are functional in a variety of different eukaryotic cell types, it would be expected that a two-hybrid assay could be performed in virtually any cell line of eukaryotic origin (e.g., insect 5 cells (SF9), fungal cells, worm cells, etc.). Other genetic systems for the detection of protein-protein interactions include the so-called SOS recruitment system (Aronheim et al., 1997). Protein-protein interactions Protein interactions are detected in various systems including the yeast two-hybrid system, 0 affinity chromatography, co-immunoprecipitation, subcellular fractionation and isolation of large molecular complexes. Each of these method is well characterized and can be readily performed by one skilled in the art. See, e.g., U.S. Patents No. 5,622,852 and 5,773,218, PCT published application No. WO 97/27296 and PCT published application No. WO 99/65939, each of which are incorporated herein by reference. 5 The protein of interest (or an interacting domain thereof) can be produced in eukaryotic or prokaryotic systems. A cDNA encoding the desired protein is introduced in an appropriate expression vector and transfected in a host cell (which could be bacteria, yeast cells, insect cells, or mammalian cells). Purification of the expressed protein is achieved by conventional biochemical and immunochemical methods well known to those skilled in the art. The purified protein is then 0 used for affinity chromatography studies: it is immobilized on a matrix and loaded on a column. Extracts from cultured cells or homogenized tissue samples are then loaded on the column in appropriate buffer, and non-binding proteins are eluted. After extensive washing, binding proteins or protein complexes are eluted using various methods such as a gradient of pH or a gradient of salt concentration. Eluted proteins can then be separated by two-dimensional gel electrophoresis, eluted 5 from the gel, and identified by micro-sequencing. The purified proteins can also be used for affinity chromatography to purify interacting proteins disclosed herein. All of these methods are well known to those skilled in the art. Similarly, both proteins of the complex of interest (or interacting domains thereof) can be produced in eukaryotic or prokaryotic systems. The proteins (or interacting domains) can be under 0 control of separate promoters or can be produced as a fusion protein. The fusion protein may include a peptide linker between the proteins (or interacting domains) which, in one embodiment, WO 00/65340 PCT/US00/10651 35 serves to promote the interaction of the proteins (or interacting domains). All of these methods are also well known to those skilled in the art. Purified proteins of interest, individually or a complex, can also be used to generate antibodies in rabbit, mouse, rat, chicken, goat, sheep, pig, guinea pig, bovine, and horse. The 5 methods used for antibody generation and characterization are well known to those skilled in the art. Monoclonal antibodies are also generated by conventional techniques. Single chain antibodies are further produced by conventional techniques. DNA molecules encoding proteins of interest can be inserted in the appropriate expression vector and used for transfection of eukaryotic cells such as bacteria, yeast, insect cells, or 0 mammalian cells, following methods well known to those skilled in the art. Transfected cells expressing both proteins of interest are then lysed in appropriate conditions, one of the two proteins is immunoprecipitated using a specific antibody, and analyzed by polyacrylamide gel electrophoresis. The presence of the binding protein (co-immunoprecipitated) is detected by immunoblotting using an antibody directed against the other protein. Co-immunoprecipitation is 5 a method well known to those skilled in the art. Transfected eukaryotic cells or biological tissue samples can be homogenized and fractionated in appropriate conditions that will separate the different cellular components. Typically, cell lysates are run on sucrose gradients, or other materials that will separate cellular components based on size and density. Subcellular fractions are analyzed for the presence of proteins of interest 0 with appropriate antibodies, using immunoblotting or immunoprecipitation methods. These methods are all well known to those skilled in the art. Disruption of protein-protein interactions It is conceivable that agents that disrupt protein-protein interactions can be beneficial in 5 many physiological disorders, including, but not-limited to NIDDM, AD and others disclosed herein. Each of the methods described above for the detection of a positive protein-protein interaction can also be used to identify drugs that will disrupt said interaction. As an example, cells transfected with DNAs coding for proteins of interest can be treated with various drugs, and co immunoprecipitations can be performed. Alternatively, a derivative of the yeast two-hybrid system, 30 called the reverse yeast two-hybrid system (Leanna and Hannink, 1996), can be used, provided that the two proteins interact in the straight yeast two-hybrid system.

WO 00/65340 PCT/US00/10651 36 Modulation of protein-protein interactions Since the interaction described herein is involved in a physiological pathway, the identification of agents which are capable of modulating the interaction will provide agents which can be used to track the physiological disorder or to use as lead compounds for development of 5 therapeutic agents. An agent may modulate expression of the genes of interacting proteins, thus affecting interaction of the proteins. Alternatively, the agent may modulate the interaction of the proteins. The agent may modulate the interaction of wild-type with wild-type proteins, wild-type with mutant proteins, or mutant with mutant proteins. Agents can be tested using transfected host cells, cell lines, cell models or animals, such as described herein, by techniques well known to those 0 of ordinary skill in the art, such as disclosed in U.S. Patents No. 5,622,852 and 5,773,218, PCT published application No. WO 97/27296 and PCT published application No. WO 99/65939, each of which are incorporated herein by reference. The modulating effect of the agent can be screened in vivo or in vitro. Exemplary of a method to screen agents is to measure the effect that the agent has on the formation of the protein complex. 5 Mutation screening The proteins disclosed in the present invention interact with one or more proteins known to be involved in a physiological pathway, such as in NIDDM or AD. Mutations in interacting proteins could also be involved in the development of the physiological disorder, such as NIDDM 0 or AD, for example, through a modification of protein-protein interaction, or a modification of enzymatic activity, modification of receptor activity, or through an unknown mechanism. Therefore, mutations can be found by sequencing the genes for the proteins of interest in patients having the physiological disorder, such as insulin, and non-affected controls. A mutation in these genes, especially in that portion of the gene involved in protein interactions in the physiological 5 pathway, can be used as a diagnostic tool, and the mechanistic understanding the mutation provides can help develop a therapeutic tool. Screening for at-risk individuals Individuals can be screened to identify those at risk by screening for mutations in the protein 0 disclosed herein and identified as described above. Alternatively, individuals can be screened by analyzing the ability of the proteins of said individual disclosed herein to form natural complexes. Further, individuals can be screened by analyzing the levels of the complexes or individual proteins WO 00/65340 PCT/US00/10651 37 of the complexes or the mRNA encoding the protein members of the complexes. Techniques to detect the formation of complexes, including those described above, are known to those skilled in the art. Techniques and methods to detect mutations are well known to those skilled in the art. Techniques to detect the level of the complexes, proteins or mRNA are well known to those skilled in the art. Cellular models of Physiological Disorders A number of cellular models of many physiological disorders or diseases have been generated. The presence and the use of these models are familiar to those skilled in the art. As an ) example, primary cell cultures or established cell lines can be transfected with expression vectors encoding the proteins of interest, either wild-type proteins or mutant proteins. The effect of the proteins disclosed herein on parameters relevant to their particular physiological disorder or disease can be readily measured. Furthermore, these cellular systems can be used to screen drugs that will influence those parameters, and thus be potential therapeutic tools for the particular physiological 5 disorder or disease. Alternatively, instead of transfecting the DNA encoding the protein of interest, the purified protein of interest can be added to the culture medium of the cells under examination, and the relevant parameters measured. Animal models ) The DNA encoding the protein of interest can be used to create animals that overexpress said protein, with wild-type or mutant sequences (such animals are referred to as "transgenic"), or animals which do not express the native gene but express the gene of a second animal (referred to as "transplacement"), or animals that do not express said protein (referred to as "knock-out"). The knock-out animal may be an animal in which the gene is knocked out at a determined time. The 5 generation of transgenic, transplacement and knock-out animals (normal and conditioned) uses methods well known to those skilled in the art. In these animals, parameters relevant to the particular physiological disorder can be measured. These parametes may include receptor function, protein secretion in vivo or in vitro, survival rate of cultured cells, concentration of particular protein in tissue homogenates, signal 0 transduction, behavioral analysis, protein synthesis, cell cycle regulation, transport of compounds across cell or nuclear membranes, enzyme activity, oxidative stress, production of pathological products, and the like. The measurements of biochemical and pathological parameters, and of WO 00/65340 PCT/US00/10651 38 behavioral parameters, where appropriate, are performed using methods well known to those skilled in the art. These transgenic, transplacement and knock-out animals can also be used to screen drugs that may influence the biochemical, pathological, and behavioral parameters relevant to the particular physiological disorder being studied. Cell lines can also be derived from these animals 5 for use as cellular models of the physiological disorder, or in drug screening. Rational drug design The goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact (e.g., agonists, antagonists, O inhibitors) in order to fashion drugs which are, for example, more active or stable forms of the polypeptide, or which, e.g., enhance or interfere with the function of a polypeptide in vivo. Several approaches for use in rational drug design include analysis of three-dimensional structure, alanine scans, molecular modeling and use of anti-id antibodies. These techniques are well known to those skilled in the art. 5 Following identification of a substance which modulates or affects polypeptide activity, the substance may be further investigated. Furthermore, it may be manufactured and/or used in preparation, i.e., manufacture or formulation, or a composition such as a medicament, pharmaceutical composition or drug. These may be administered to individuals. A substance identified as a modulator of polypeptide function may be peptide or non-peptide 0 in nature. Non-peptide "small molecules" are often preferred for many in vivo pharmaceutical uses. Accordingly, a mimetic or mimic of the substance (particularly if a peptide) may be designed for pharmaceutical use. The designing of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a "lead" compound. This approach might .5 be desirable where the active compound is difficult or expensive to synthesize or where it is unsuitable for a particular method of administration, e.g., pure peptides are unsuitable active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal. Mimetic design, synthesis and testing is generally used to avoid randomly screening large numbers of molecules for a target property. 0 Once the pharmacophore has been found, its structure is modeled according to its physical properties, e.g., stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g., spectroscopic techniques, x-ray diffraction data and NMR. Computational analysis, similarity WO 00/65340 PCT/US00/10651 39 mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modeling process. A template molecule is then selected, onto which chemical groups that mimic the pharmacophore can be grafted. The template molecule and the chemical groups grafted thereon can 5 be conveniently selected so that the mimetic is easy to synthesize, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound. Alternatively, where the mimetic is peptide-based, further stability can be achieved by cyclizing the peptide, increasing its rigidity. The mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent it is exhibited. 0 Further optimization or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing. Diagnostic Assays The identification of the interactions disclosed herein enables the development of diagnostic 5 assays and kits, which can be used to determine a predisposition to or the existence of a physiological disorder. In one aspect, one of the proteins of the interaction is used to detect the presence of a "normal" second protein (i.e., normal with respect to its ability to interact with the first protein) in a cell extract or a biological fluid, and further, if desired, to detect the quantitative level of the second protein in the extract or biological fluid. The absence of the "normal" second protein 0 would be indicative of a predisposition or existence of the physiological disorder. In a second aspect, an antibody against the protein complex is used to detect the presence and/or quantitative level of the protein complex. The absence of the protein complex would be indicative of a predisposition or existence of the physiological disorder. Z5 EXAMPLES The present invention is further detailed in the following Examples, which are offered by way of illustration and are not intended to limit the invention in any manner. Standard techniques well known in the art or the techniques specifically described below are utilized.

WO 00/65340 PCT/US00/10651 40 EXAMPLE 1 Yeast Two-Hybrid System The principles and methods of the yeast two-hybrid systems have been described in detail (Bartel and Fields, 1997). The following is thus a description of the particular procedure that we used, which was applied to all proteins. The cDNA encoding the bait protein was generated by PCR from brain cDNA. Gene specific primers were synthesized with appropriate tails added at their 5' ends to allow recombination into the vector pGBTQ. The tail for the forward primer was 5' GCAGGAAACAGCTATGACCATACAGTCAGCGGCCGCCACC-3' (SEQ ID NO: 1) and the tail for the reverse ) primer was 5'-ACGGCCAGTCGCGTGGAGTGTTATGTCATGCGGCCGCTA-3' (SEQ ID NO:2). The tailed PCR product was then introduced by recombination into the yeast expression vector pGBTQ, which is a close derivative of pGBTC (Bartel et al., 1996) in which the polylinker site has been modified to include M13 sequencing sites. The new construct was selected directly in the yeast J693 for its ability to drive tryptophane synthesis (genotype of this strain: Mat c, ade2, his3, leu2, trpl, 5 URA3::GAL1-lacZ LYS2::GAL1-HIS3 gal4del gal80del cyhR2). In these yeast cells, the bait is produced as a C-terminal fusion protein with the DNA binding domain of the transcription factor Gal4 (amino acids 1 to 147). A total human brain (37 year-old male Caucasian) cDNA library cloned into the yeast expression vector pACT2 was purchased from Clontech (human brain MATCHMAKER cDNA, cat. # HL4004AH), transformed into the yeast strain J692 (genotype of ) this strain: Mat a, ade2, his3, leu2, trpl, URA3::GAL1-lacZ LYS2::GAL1-HIS3 gal4del gal80del cyhR2), and selected for the ability to drive leucine synthesis. In these yeast cells, each cDNA is expressed as a fusion protein with the transcription activation domain of the transcription factor Gal4 (amino acids 768 to 881) and a 9 amino acid hemagglutinin epitope tag. J693 cells (Mat ca type) expressing the bait were then mated with J692 cells (Mat a type) expressing proteins from the 5 brain library. The resulting diploid yeast cells expressing proteins interacting with the bait protein were selected for the ability to synthesize tryptophane, leucine, histidine, and P-galactosidase. DNA was prepared from each clone, transformed by electroporation into E. coli strain KC8 (Clontech KC8 electrocompetent cells, cat # C2023-1), and the cells were selected on ampicillin-containing plates in the absence of either tryptophane (selection for the bait plasmid) or leucine (selection for 0 the brain library plasmid). DNA for both plasmids was prepared and sequenced by di deoxynucleotide chain termination method. The identity of the bait eDNA insert was confirmed and the cDNA insert from the brain library plasmid was identified using BLAST program against public WO 00/65340 PCT/US00/10651 41 nucleotides and protein databases. Plasmids from the brain library (preys) were then individually transformed into yeast cells together with a plasmid driving the synthesis of lamin fused to the Gal4 DNA binding domain. Clones that gave a positive signal after P-galactosidase assay were considered false-positives and discarded. Plasmids for the remaining clones were transformed into yeast cells 5 together with plasmid for the original bait. Clones that gave a positive signal after P-galactosidase assay were considered true positives. EXAMPLE 2 Identification of Glut4/CARP Interaction 0 A yeast two-hybrid system as described in Example 1 using amino acids 463-509 of Glut4 (Swiss Protein (SP) accession no. P 14672) as bait was performed. One clone that was identified by this procedure included the amino acids encoded by nucleotides 312-1155 of CARP (GenBank (GB) accession no. X83703). 5 EXAMPLE 3 Identification of GLUT1/DRAL(FHL2) Interaction A yeast two-hybrid system as described in Example 1 using amino acids 448-492 of Glutl1 (SP accession no. P11166) as bait was performed. One clone that was identified by this procedure included amino acids 1-279 of DRAL(FHL2) (SP accession no. Q13229). 0 EXAMPLE 4 Identification of GLUT1/Myosin Heavy Chain Interaction A yeast two-hybrid system as described in Example 1 using amino acids 448-492 of Glutl (SP accession no. P11166) as bait was performed. One clone that was identified by this procedure !5 included amino acids 1589-1909 of myosin heavy chain (SP accession no. P13533). EXAMPLE 5 Identification of GLUT1/HSS Interaction A yeast two-hybrid system as described in Example 1 using amino acids 448-492 of Glutl 30 (SP accession no. P11166) as bait was performed. One clone that was identified by this procedure included amino acids encoded by nucleotides 1-? of HSS (GB accession no. X91879).

WO 00/65340 PCT/US00/10651 42 EXAMPLE 6 Identification of OGTase/Mvosin Heavy Chain Interaction A yeast two-hybrid system as described in Example 1 using amino acids encoded by nucleotides 1016-1349 of OGTase (GB accession no. U77413) as bait was performed. One clone 5 that was identified by this procedure included amino acids encoded by nucleotides 1773-2283 of myosin heavy chain (GB accession no. AF 111785). EXAMPLE 7 Identification of IRAP/14-3-3 beta Interaction 0 A yeast two-hybrid system as described in Example 1 using amino acids encoded by nucleotides 449-880 of IRAP (GB accession no. U62768) as bait was performed. One clone that was identified by this procedure included amino acids 97-236 of 14-3-3 beta (SP accession no. P31946). 5 EXAMPLE 8 Identification ofl IRAP/HSS Interaction A yeast two-hybrid system as described in Example 1 using amino acids encoded by nucleotides 62-880 of IRAP (GB accession no. U62768) as bait was performed. One clone that was identified by this procedure included amino acids encoded by nucleotides 726-1446 of HSS (GB 0 accession no. X91879). EXAMPLE 9 Identification of PI-3K110/Complement Protein C4 Interaction A yeast two-hybrid system as described in Example 1 using amino acids 1-300 of PI-3 .5 kinase p110 subunit (SP accession no. P42338) as bait was performed. One clone that was identified by this procedure included amino acids 1056-1277 of complementj protein C4 (SP accession no. P01028). EXAMPLE 10 0 Identification of PI-3K110/Tenascin XB Interaction A yeast two-hybrid system as described in Example 1 using amino acids 1-300 of PI-3 kinase p 1 10 subunit (SP accession no. P42338) as bait was performed. One clone that was WO 00/65340 PCT/US00/10651 43 identified by this procedure included amino acids 3573-3787 of tenascin XB (SP accession no. P78530). EXAMPLE 11 5 Identification of PI-3K110/GAA Interaction A yeast two-hybrid system as described in Example 1 using amino acids 1-300 of PI-3 kinase p110 subunit (SP accession no. P42338) as bait was performed. One clone that was identified by this procedure included amino acids 513-? of GAA (SP accession no. P10253). 0 EXAMPLE 12 Identification of MM- 1/C-Nap 1 Interaction A yeast two-hybrid system as described in Example 1 using amino acids 27-175 of MM-1 (SP accession no. Q99471) as bait was performed. One clone that was identified by this procedure included amino acids encoded by nucleotides 1508-1949 of C-Napl (GB accession no. AF049105). 5 EXAMPLE 13 Identification of MM-i1/Beta Spectrin Interaction A yeast two-hybrid system as described in Example 1 using amino acids 1-175 of MM-1 (SP accession no. Q99471) as bait was performed. One clone that was identified by this procedure 0 included amino acids 1545-1789 of beta spectrin (SP accession no. Q01082). EXAMPLE 14 Identification of MM- 1/KIAA0477 Interaction A yeast two-hybrid system as described in Example 1 using amino acids 27-175 of MM-1 5 (SP accession no. Q99471) as bait was performed. One clone that was identified by this procedure included amino acids encoded by nucleotides 2448-3207 of KIAA0477 (GB accession no. AB007946). EXAMPLE 15 .0 Identification of Dynamin/CALM Interaction A yeast two-hybrid system as described in Example 1 using amino acids 250-700 of dynamin (SP accession no. Q05193) as bait was performed. One clone that was identified by this WO 00/65340 PCT/US00/10651 44 procedure included amino acids encoded by nucleotides 948-1599 of CALM (GB accession no. U45976). EXAMPLE 16 5 Identification of Dvnamin/Psme3 Interaction A yeast two-hybrid system as described in Example 1 using amino acids 250-700 of dynamin (SP accession no. Q05193) as bait was performed. One clone that was identified by this procedure included amino acids encoded by nucleotides 378-966 of Psme3 (GB accession no. U11292). 0 EXAMPLE 17 Identification of Naflb/I-TRAF Interaction A yeast two-hybrid system as described in Example 1 using amino acids encoded by nucleotides 590-1610 ofNaflb (GB accession no. Q05193) as bait was performed. One clone that 5 was identified by this procedure included amino acids encoded by nucleotides 209-1420 of I-TRAF (GB accession no. U59863). EXAMPLE 18 Identification of Akt 1l/NuMA1 Interaction 0 A yeast two-hybrid system as described in Example 1 using amino acids 1-118 of Aktl (SP accession no. P31749) as bait was performed. One clone that was identified by this procedure included amino acids encoded by nucleotides 813-1341 of NuMAl (GB accession no. Z11584). EXAMPLE 19 5 Identification of Akt2/NuMA 1 Interaction A yeast two-hybrid system as described in Example 1 using amino acids 1-152 of Akt2 (SP accession no. P31751) as bait was performed. One clone that was identified by this procedure included amino acids encoded by nucleotides 552-1353 ofNuMAl (GB accession no. ZI 1584).

WO 00/65340 PCT/US00/10651 45 EXAMPLE 20 Identification of Akt2/BAP31 I Interaction A yeast two-hybrid system as described in Example 1 using amino acids encoded by nucleotides 915-1311 of Akt2 (GB accession no. M95936) as bait was performed. One clone that 5 was identified by this procedure included amino acids encoded by nucleotides 469-877 of BAP31 (GB accession no. NM00574). EXAMPLE 21 Identification of Akt2/Beta Adaptin Interaction 0 A yeast two-hybrid system as described in Example 1 using amino acids encoded by nucleotides 915-1311 of Akt2 (GB accession no. M95936) as bait was performed. One clone that was identified by this procedure included amino acids 214-594 of beta adaptin (SP accession no. P21851). 5 EXAMPLE 22 Identification of OGTase/Desmin Interaction A yeast two-hybrid system as described in Example 1 using amino acids encoded by nucleotides 1016-1349 of OGTase (GB accession no. U77413) as bait was performed. One clone that was identified by this procedure included amino acids 219-449 of desmin (SP accession no. 0 P17661). EXAMPLE 23 Identification of OGTase/Alpha-karyopherin Interaction A yeast two-hybrid system as described in Example 1 using amino acids encoded by 5 nucleotides 1016-1349 of OGTase (GB accession no. U77413) as bait was performed. One clone that was identified by this procedure included amino acids 152-444 of alpha-karyopherin (SP accession no. P52294). EXAMPLE 24 0 Identification of OGTase/Glutaminvl tRNA Synthetase Interaction A yeast two-hybrid system as described in Example 1 using amino acids encoded by nucleotides 1016-1349 of OGTase (GB accession no. U77413) as bait was performed. One clone WO 00/65340 PCT/US00/10651 46 that was identified by this procedure included amino acids 36-161 of glutaminyl tRNA synthetase (SP accession no. P47897). EXAMPLE 25 5 Identification of OGTase/Clone 25100 Interaction A yeast two-hybrid system as described in Example 1 using amino acids encoded by nucleotides 1016-1349 of OGTase (GB accession no. U77413) as bait was performed. One clone that was identified by this procedure included amino acids 11-338 of clone 25100 (GB accession no. AF131780). ) EXAMPLE 26 Identification of PTP 1 b/VAP-A Interaction A yeast two-hybrid system as described in Example 1 using amino acids 280-436ofPTPlb (SP accession no. P18031) as bait was performed. One clone that was identified by this procedure 5 included amino acids encoded by nucleotides 9-? of VAP-A (GB accession no. AF086627). EXAMPLE 27 Identification of Rab4/Alpha-catenin-like Protein Interaction A yeast two-hybrid system as described in Example 1 using amino acids 1-214 of Rab4 (SP S accession no. P20338) as bait was performed. One clone that was identified by this procedure included amino acids encoded by nucleotides 1573-1987 of alpha-catenin-like protein (GB accession no. U97067). EXAMPLE 28 5 Identification of Rab4/Rab2 Interaction A yeast two-hybrid system as described in Example 1 using amino acids 1-214 of Rab4 (SP accession no. P20338) as bait was performed. One clone that was identified by this procedure included amino acids 27-177 of Rab2 (SP accession no. P08886).

WO 00/65340 PCT/US00/10651 47 EXAMPLE 29 Identification of Glut4/PN7065 Interaction A yeast two-hybrid system as described in Example 1 using amino acids 463-509 of Glut4 (Swiss Protein (SP) accession no. P14672) as bait was performed. One clone that was identified by 5 this procedure included novel protein fragment PN7065. The DNA sequence and the predicted protein sequence for PN7065 are set forth in 75 and 76, respetively. TABLE 75 Nucleotide Sequence of PN7065 (SEQ ID NO:3) 0 CGCCGGTGGATGCGGGCTGAGCCCTGCTTGCCGGGACCCGCCTGCCCCGCCTTCTCCGCA CACAGCTACACCTCCAACCTGGGCGACTACGATGAGCAGGCGCTGGGTATCATGCAGACC CTGGGCGTGGACCGGCAGAGGACGGTGGAGTCACTGCAAAACAGCAGCTATAACCACTTT GCTGCCATTTATTACCTCCTCCTTGAGCGGCTCAAGGAGTATCGGAATGCCCAGTGCGCC CGCCCCGGGCCTGCCAGGCAGCCGCGGCCTCGGAGCTCGGACCTCAGTGGTTTGGAGGTG 5 CCTCAGGAAGGTCTTTCCACCGACCCTTTCCGACCTGCCTTGCTGTGCCCGCAGCCGCAG ACCTTGGTGCAGTCCGTCCTCCAGGCCGAGATGGACTGTGGGCTCCAGAGCTCGCTGCAG TGGCCCTTGTTCTTCCCGGTGGATGCCAGCTGCAGCGGAGTGTTCCGGCCCCGGCCCGTG TCCCCAAGCAGCCTGCTGGACACAGCCATCAGTGAGGAGGCCAGGCAGGGGCCGGGCCTA GAGGAGGAGCAGGACACGCAGGAGTCCCTGCCCAGCAGCACGGGCCGGGGGCACACCCTG 0 GCCGAGGTCTCCACCCGCCTCTCCCCACTCACCGCGCCAG TABLE 76 Predicted Protein Sequence of PN7065 (SEQ ID NO:4) RRWMRAEPCLPGPACPAFSAHSYTSNLGDYDEQALGIMQTLGVDRQRTVESLQNSSYNHF 5 AAIYYLLLERLKEYRNAQCARPGPARQPRPRSSDLSGLEVPQEGLSTDPFRPALLCPQPQ TLVQSVLQAEMDCGLQSSLQWPLFFPVDASCSGVFRPRPVSPSSLLDTAISEEARQGPGL EEEQDTQESLPSSTGRGHTLAEVSTRLSPLTAP EXAMPLE 30 0 Identification of Glut4/PN7386 Interaction A yeast two-hybrid system as described in Example 1 using amino acids 478-509 of Glut4 (SP accession no. P 14672) as bait was performed. One clone that was identified by this procedure included novel protein fragment PN7386. The DNA sequence and the predicted protein sequence for PN7386 are set forth in Tables 77 and 78, respectively. 5 WO 00/65340 PCT/US00/10651 48 TABLE 77 Nucleotide Sequence of PN7386 (SEQ ID NO:5) GGCCAATGTTGACGTTTTGGTAGGCTATGCAGACATCCATGGAGACTTACTACCTATAAA TAATGATGATAATTATCACAAAGCTGTTTCAACGGCCAATCCACTGCTTAGGATATTTAT 5 ACAAAAGAAGGAAGAAGCAGACTACAGTGCCTTTGGTACAGACACGCTAATAAAGAAGAA GAATGTTTTAACCAACGTATTGCGTCCTGACAACCATAGAAAAAAGCCACATATAGTCAT TAGTATGCCCCAAGACTTTAGACCTGTGTCTTCTATTATAGACGTGGATATTCTCCCAGA AACGCATCGTAGGGTACGTCTTTACAAATACGGCACGGAGAAACCCCTAGGATTCTACAT CCGGGATGGCTCCAGTGTCAGGGTAACACCACATGGCTTAGAAAAGGTTCCAGGGATCTT 0 TATATCCAGGCTTGTCCCAGGAGGTCTGGCTCAAAGTACAGGACTATTAGCTGTTAATGA TGAAGTTTTAGAAGTTAATGGCATAGAAGTTTCAGGGAAGAGCCTTGATCAAGTAACAGA CATGATGATTGCAAATAGCCGTAACCTCATCATAACAGTGAGACCGGCAAACCAGAGGAA TAATGTTGTGAGGAACAGTCGGACTTCTGGCAGTTCCGGTCAGTCTACTGATAACAGCCT TCTTGGCTACCCACAGCAGATTGAACCAAGCTTTGAGCCAGAGGATGAAGACAGCGAAGA 5 AGATGACATTATCATTGAAGACAATGGAGTGCCACAGCAGATTCCAAAAGCTGTTCCTAA TACTGAGAGCCTGGAGTCATTAACACAGATAGAGCTAAGCTTTGAGTCTGGACAGAATGG CTTTATTCCCTCTAATGAAGTGAGCTTAGCAGCCATAGCAAGCAGCTCAAACACGGAATT TGAAACACATGCTCCAGATCAAAAACTCTTAGAAGAAGATGGAACAATCATAACATTATG AAACCGTGGTTTGAATGTTTTCAGAGTGAGGATGCCATGAGGACTTGTACATTTGGCTAG 0 TTTAGGCCAATGTTGACGTTTTGGTAGGCTATGCAGACATCCATG TABLE 78 Predicted Amino Acid Sequence of PN7386 (SEQ ID NO:6) ANVDVLVGYADIHGDLLPINNDDNYHKAVSTANPLLRIFIQKKEEADYSAFGTDTLIKKK 5 NVLTNVLRPDNHRKKPHIVISMPQDFRPVSSIIDVDILPETHRRVRLYKYGTEKPLGFYI RDGSSVRVTPHGLEKVPGIFI SRLVPGGLAQSTGLLAVNDEVLEVNGIEVSGKSLDQVTD MMIANSRNLIITVRPANQRNNVVRNSRTSGSSGQSTDNSLLGYPQQIEPSFEPEDEDSEE DDIIIEDNGVPQQIPKAVPNTESLESLTQIELSFESGQNGFIPSNEVSLAAIASSSNTEF ETHAPDQKLLEEDGT IITL 0 EXAMPLE 31 Identification of OGTase/PN6931 Interaction A yeast two-hybrid system as described in Example 1 using amino acids encoded by nucleotides 1016-1618 of OGTase (GenBank (GB) accession no. U77413) as bait was performed. 5 One clone that was identified by this procedure included novel protein fragment PN6931. The DNA sequence and the predicted protein sequence for PN6931 are set forth in Tables 79 and 80, respectively. TABLE79 0 Nucleotide Sequence of PN6931 (SEQ ID NO:7)

AGGGAGGAGAAGCTGAGCCAGGATGAGATCGTGCTGGGCACCAAGGCTGTCATCCAGGGA

WO00/65340 PCTUS00/10651 49 CTGGAGACTCTGCGTGGGGAGCATCGTGCCCTGCTGGCTCCTCTGGTTGCACCTGAGGCC GGCGAAGCCGAGCCTGGCTCGCAGGAGCGCTGCATCCTCCTGCGTCGCTCCCTGGAAGCC ATTGAGCTTGGGCTGGGGGAGGCCCAGGTGATCTTGGCATTGTCGAGCCACCTGGGGGCT GTAGAATCAGAGAAGCAGAAGCTGCGGGCGCAGGTGCGGCGTCTGGTGCAGGAGAACCAG 5 TGGCTGCGTGAGGAGCTGCCGGGGACACAGCAKAAGCTGCAGCGCAGTGAGCAGGCCGTG GCCCAGCTCGAGGAGGAGAAGCAGCACTTGCTGTTCATGARCCAGATCCGCAGTTGGATG AAGACGCCTYCCCTAACGAGGAGAAGGGGGACGTCCCCAAAGACACACTGGATGACCTGT TCCCCAATGAGGATGAGCAGAGCCCAGCCCCTAGCCCAGGAGGAGGGGATGTGTCTGGTC AGCATGGGGGATACGAGATCCCGGCCCGGCTCCGCACCCTGCACAACTGGTGATCCAATA 0 CGCCTCACAGGGCCGCTACGAGGTAGCTGTGCCACTCTGCAAGCAGGCACTCGAAGACTG GAGAAGACGTCAGGCCACGACCACCCTGACGTTGCCACCATGCTGAACATCCTGGCACTG GTCTATCGGGATCAGAACAAGTACAAGGAGGCTGCCCACCTGCTCAATGATGCTCTGGCC ATCCGGGAGAAAACACTGGGCAAGGACCACCCAGCCGTGGCTGCGACACTAAACAACCTG GCAGTCCTGTATAGCGCAGAG 5 TABLE80 Predicted Protein Sequence of PN6931 (SEQ ID NO:8) REEKLSQDEIVLGTKAVIQGLETLRGEHRALLAPLVAPEAGEAEPGSQERCILLRRSLEA IELGLGEAQVILALSSHLGAVESEKQKLRAQVRRLVQENQWLREELPGTQXKLQRSEQAV 0 AQLEEEKQHLLFMXQIRSWMKTPXLTRRRGTSPKTHWMTCSPMRMSRAQPLAQEEGMCLV SMGDTRSRPGSAPCTTGDPIRLTGPLRGSCATLQAGTRRLEKTSGHDHPDVATMLNILAL VYRDQNKYKEAAHLLNDALAIREKTLGKDHPAVAATLNNLAVLYSAE EXAMPLE 32 5 Identification of Naflb/PN7582 Interaction A yeast two-hybrid system as described in Example 1 using amino acids encoded by nucleotides 233-1568 ofNaflb (GB accession no. AJ011896) as bait was performed. One clone that was identified by this procedure included novel protein fragment PN7582. The DNA sequence and the predicted protein sequence for PN7582 are set forth in Tables 81 and 82, respectively. 0 TABLE 81 Nucleotide Sequence for PN7582 (SEQ ID NO:9) ATTTCAAGGGGGCTGCTGTACCCCCAGGCATGTGTCTGTATATCGCACAGGAAGAAGGAA AGTAAGGACATTGCCAGCAAATATCTTACATCTCATCAGCCTATACTGTGTCTCCTGACC 5 ACTCCTAACTGCAAAGGATGCTGGGAAAAAAAGAGCATTGTAGCTTTTCCAGCCTCTGTG GTAGGCGCAGATAAGGGATTAGAGTTGGGTGTTACTGAATCAATGTATCAGACACTTCTC AGTCAGGCTAGAGCCAGATTTAACTAGATTTAGCAGGAAAAGTATGTTTCTTTCACCTGC ATGTAATGAAGGAAATCTATGTCCTTCATACACTTAATAAACCTGTAAGTCTCTACTATG GGCAGGTACTGTGCTAGCTAGACATTACAATGTGTGGGGGCAGACACAAAGATGGGAACA 0 GTAGACACTGGGGAACCCTAGAGGGGGGAGGTTGGGAGTAGGGGAAGGGTTGAAAAATGA TTGGGTACTATGCTCACTACCTGGGTGATGGGATCATTTGTACACCAAATGCCAGCAACA CACAATTTACCCGTGTAACACACCGGCACGTGTACCCCCTGAACCTAAGATGAAAGCCGA

A

WO 00/65340 PCT/US00/10651 50 TABLE 82 Predicted Amino Acid Sequence for PN7582 (SEQ ID NO:10) ISRGLLYPQACVCISHRKKESKDIASKYLTSHQPILCLLTTPNCKGCWEKKSIVAFPASV 5 VGADKGLELGVTESMYQTLLSQARARFN EXAMPLE 33 Identification of OGTase/Talin Interaction A yeast two-hybrid system as described in Example 1 using amino acids encoded by 0 nucleotides 1016-1349 of OGTase (Genbank (GB) accession no. U77413) as bait was performed. One clone that was identified by this procedure included amino acids 812 et seq. of talin (GB accession no. AF078828). EXAMPLE 34 5 Identification of OGTase/MOP2 Interaction A yeast two-hybrid system as described in Example 1 using amino acids encoded by nucleotides 1016-1349 of OGTase (Genbank (GB) accession no. U77413) as bait was performed. One clone that was identified by this procedure included amino acids 397-546 of MOP2 (Swiss Protein (SP) accession no. Q99814). 0 EXAMPLE 35 Identification of OGTase/Clone 25100 Interaction A yeast two-hybrid system as described in Example 1 using amino acids encoded by nucleotides 1016-1349 of OGTase (Genbank (GB) accession no. U77413) as bait was performed. !5 One clone that was identified by this procedure included amino acids encoded by nucleotides 31 1014 of clone 25100 (GB accession no. AF121780). EXAMPLE 36 Identification of OGTase/KIAA0443 Interaction 30 A yeast two-hybrid system as described in Example 1 using amino acids encoded by nucleotides 1016-1349 of OGTase (Genbank (GB) accession no. U77413) as bait was performed.

WO 00/65340 PCT/US00/10651 51 One clone that was identified by this procedure included amino acids encoded by nucleotides 3468 4106 of KIAA0443 (GB accession no. AB007903). EXAMPLE 37 5 Identification of OGTase/EGR1 Interaction A yeast two-hybrid system as described in Example 1 using amino acids encoded by nucleotides 1016-1349 of OGTase (Genbank (GB) accession no. U77413) as bait was performed. One clone that was identified by this procedure included amino acids 415-544 of EGR1 (SP accession no. P18146). 0 EXAMPLE 38 Identification of OGTase/Dynamin II Interaction A yeast two-hybrid system as described in Example 1 using amino acids encoded by nucleotides 1016-1349 of OGTase (Genbank (GB) accession no. U77413) as bait was performed. 5 One clone that was identified by this procedure included amino acids 515-823 of dynamin II (SP accession no. P50570). EXAMPLE 39 Identification of OGTase/INT-6 Interaction 10 A yeast two-hybrid system as described in Example 1 using amino acids encoded by nucleotides 1016-1349 of OGTase (Genbank (GB) accession no. U77413) as bait was performed. One clone that was identified by this procedure included amino acids encoded by nucleotides 824 1191 of Int-6 (GB accession no. U62962). :5 EXAMPLE 40 Identification of OGTase/HSPCO28 Interaction A yeast two-hybrid system as described in Example 1 using amino acids encoded by nucleotides 1016-1349 of OGTase (Genbank (GB) accession no. U77413) as bait was performed. One clone that was identified by this procedure included amino acids encoded by nucleotides 58-443 )0 of HSPCO28 (GB accession no. AF083246).

WO 00/65340 PCT/US00/10651 52 EXAMPLE 41 Identification of OGTase/BAP31 Interaction A yeast two-hybrid system as described in Example 1 using amino acids encoded by nucleotides 1016-1349 of OGTase (Genbank (GB) accession no. U77413) as bait was performed. 5 One clone that was identified by this procedure included amino acids 116-215 of BAP31 (SP accession no. P51572). EXAMPLE 42 Identification of OGTase/Interferon-Ind Protein Interaction 0 A yeast two-hybrid system as described in Example 1 using amino acids encoded by nucleotides 1016-1349 of OGTase (Genbank (GB) accession no. U77413) as bait was performed. One clone that was identified by this procedure included amino acids 1 et seq. of interferon-ind protein (SP accession no. P09913). 5 EXAMPLE 43 Identification of Glut4/Beta-catenin Interaction A yeast two-hybrid system as described in Example 1 using amino acids 463-509 of Glut4 (Swiss Protein (SP) accession no. P14672) as bait was performed. One clone that was identified by this procedure included amino acids 579-782 of Beta-catenin (SP accession no. P35222). :0 EXAMPLE 44 Identification of Glut4/Alpha-SNAP Interaction A yeast two-hybrid system as described in Example 1 using amino acids 463-509 of Glut4 (SP accession no. P14672) as bait was performed. One clone that was identified by this procedure .5 included amino acids 36-241 of Alpha-SNAP (SP accession no. P54920). EXAMPLE 45 Identification of Glut4/MAPKKK6 Interaction A yeast two-hybrid system as described in Example 1 using amino acids 463-509 of Glut4 30 (SP accession no. P14672) as bait was performed. One clone that was identified by this procedure included amino acids 824-1012 of MAPKKK6 (GenBank (GB) accession no. AF 100318).

WO 00/65340 PCT/US00/10651 53 EXAMPLE 46 Identification of GLUT4/Tropomyosin 3 Interaction A yeast two-hybrid system as described in Example 1 using amino acids 434-509 of Glut4 (SP accession no. P 14672) as bait was performed. One clone that was identified by this procedure 5 included amino acids 171-286 of tropomyosin 3 (SP accession no. P06753). EXAMPLE 47 Identification of GLUT 1/DRAL/FHL2 Interaction A yeast two-hybrid system as described in Example 1 using amino acids 448-492 of Glutl1 0 (SP accession no. P11166) as bait was performed. One clone that was identified by this procedure included amino acids 1-280 of DRAL/FHL2 (SP accession no. Q13229). EXAMPLE 48 Identification of GLUT1/MYSA Interaction 5 A yeast two-hybrid system as described in Example 1 using amino acids 448-492 of Glutl (SP accession no. P11166) as bait was performed. One clone that was identified by this procedure included amino acids 1589-1909 of MYSA (SP accession no. P13533). EXAMPLE 49 0 Identification of IRAP/SG2NA Interaction A yeast two-hybrid system as described in Example 1 using amino acids encoded by nucleotides 2311-3136 of IRAP (GB accession no. U62768) as bait was performed. One clone that was identified by this procedure included amino acids 623-713 of SG2NA (SP accession no. P70483). '5 EXAMPLE 50 Identification of IRAP/SLAP-2 Interaction A yeast two-hybrid system as described in Example 1 using amino acids encoded by nucleotides 62-880 of IRAP (GB accession no. U62768) as bait was performed. One clone that was .0 identified by this procedure included amino acids 236-453 of SLAP-2 (GB accession no. AF100750).

WO 00/65340 PCT/US00/10651 54 EXAMPLE 51 Identification of OGTase/14-3-3 Epsilon Interaction A yeast two-hybrid system as described in Example 1 using amino acids encoded by nucleotides 1016-1618 of OGTase (GB accession no. U77413) as bait was performed. One clone 5 that was identified by this procedure included amino acids 21-232 of 14-3-3 epsilon (SP accession no. P29360). EXAMPLE 52 Identification of PI-3K85/Chromogranin Interaction 0 A yeast two-hybrid system as described in Example 1 using amino acids 1-250 of PI-3 kinase p85 subunit (SP accession no. P27986) as bait was performed. One clone that was identified by this procedure included amino acids 2-322 of chromogranin (SP accession no. P13521). EXAMPLE 53 5 Identification of PI-3K85/SLP-76 Interaction A yeast two-hybrid system as described in Example 1 using amino acids 320-440 of PI-3 kinase p85 subunit (SP accession no. P27986) as bait was performed. One clone that was identified by this procedure included amino acids 291-486 of SLP-76 (GB accession no. U20158). 20 EXAMPLE 54 Identification of PI-3K85/SLP-76 Interaction A yeast two-hybrid system as described in Example 1 using amino acids 600-724 of PI-3 kinase p85 subunit (SP accession no. P27986) as bait was performed. One clone that was identified by this procedure included amino acids 291-486 of SLP-76 (GB accession no. U20158). 25 EXAMPLE 55 Identification of PI-3K85/14-3-3-zeta Interaction A yeast two-hybrid system as described in Example 1 using amino acids 425-630 of PI-3 kinase p85 subunit (SP accession no. P27986) as bait was performed. One clone that was identified 30 by this procedure included amino acids 79-246 of 14-3-3-zeta (AP accession no. P29213).

WO 00/65340 PCT/US00/10651 55 EXAMPLE 56 Identification of PI-3K85/14-3-3-eta Interaction A yeast two-hybrid system as described in Example 1 using amino acids 425-630 of PI-3 kinase p85 subunit (SP accession no. P27986) as bait was performed. One clone that was identified 5 by this procedure included amino acids beginning with residue 91 of 14-3-3-eta (SP accession no. Q04917). EXAMPLE 57 Identification of PI-3K85/TACC2 Interaction 0 A yeast two-hybrid system as described in Example 1 using amino acids 600-724 of PI-3 kinase p85 subunit (SP accession no. P27986) as bait was performed. One clone that was identified by this procedure included amino acids 350-600 of TACC2 (GB accession no. AF095791). EXAMPLE 58 5 Identification of Glut4/MM-1 Interaction A yeast two-hybrid system as described in Example 1 using amino acids 463-509 of Glut4 (Swiss Protein (SP) accession no. P14672) as bait was performed. One clone that was identified by this procedure included amino acids 27-168 of MM-1 (GenBank (GB) accession no. D89667). 10 EXAMPLE 59 Identification of Glut 1 /KIAA0144 Interaction A yeast two-hybrid system as described in Example 1 using amino acids 463-492 of Glutl (SP accession no. P11166) as bait was performed. One clone that was identified by this procedure included amino acids beginning with residue 691 of KIAA0144 (GB accession no. D63478). 25 EXAMPLE 60 Identification of Glutl/Dynamin Interaction A yeast two-hybrid system as described in Example 1 using amino acids 463-492 of Glutl (Swiss. Protein (SP) accession no. P11166) as bait was performed. One clone that was identified by 30 this procedure included amino acids 104-365 of dynamin (GB accession no. L07807).

WO 00/65340 PCT/US00/10651 56 EXAMPLE 61 Identification of Glut 1/Clone 25204 Interaction A yeast two-hybrid system as described in Example 1 using amino acids 463-492 of Glutl (Swiss Protein (SP) accession no. P11166) as bait was performed. One clone that was identified by 5 this procedure included undetermined amino acids residues of Clone 25204 (GB accession no. AF 131749). EXAMPLE 62 Identification of IRAP/VAP-A Interaction 0 A yeast two-hybrid system as described in Example 1 using amino acids encoded by nucleotides 62-880 of IRAP (GB accession no. U62768) as bait was performed. One clone that was identified by this procedure included amino acids 3-243 ofVAP-A (GB accession no. AF086627). EXAMPLE 63 5 Identification of OGTase/Nafl a Interaction A yeast two-hybrid system as described in Example 1 using amino acids encoded by nucleotides 1016-1618 of OGTase (GB accession no. U77413) as bait was performed. One clone that was identified by this procedure included amino acids 41-486 of Nafla (GB accession no. AJ0 11895). :0 EXAMPLE 64 Identification of OGTasePP5/Alpha-2-Catenin A yeast two-hybrid system as described in Example 1 using acids encoded by nucleotides 1016-1618 of OGTase (GB accession no. U77413) as bait was performed. One clone that was .5 identified by this procedure included amino acids 366-469 of Alpha-2-Catenin (GB accession no. M94151). EXAMPLE 65 Identification of PI-3K110/TRIP 15 Interaction 30 A yeast two-hybrid system as described in Example 1 using amino acids 1-300 of PI-3K110 (SP accession no. P42338) as bait was performed. One clone that was identified by this procedure included amino acids 24-233 of TRIP15 (GB accession no. L40388).

WO 00/65340 PCT/US00/10651 57 EXAMPLE 66 Identification of Glut4/14-3-3 Zeta Interaction A yeast two-hybrid system as described in Example 1 using amino acids 463-509 of Glut4 5 (Swiss Protein (SP) accession no. P14672) as bait was performed. Three clones that were identified by this procedure included amino acids 1-121, 1-200 and 1-209 of 14-3-3 zeta (SP accession no. P29213). EXAMPLE 67 3 Identification of Glut4/KIAA0282 Interaction A yeast two-hybrid system as described in Example 1 using amino acids 463-509 of Glut4 (Swiss Protein (SP) accession no. P14672) as bait was performed. One clone that was identified by this procedure included amino acids 229-379 of KIAA0282 (GenBank (GB) accession no. D87458), an efp-like protein. 5 EXAMPLE 68 Identification of Glut4/Tankyrase Interaction A yeast two-hybrid system as described in Example 1 using amino acids 463-509 of Glut4 (Swiss Protein (SP) accession no. P14672) as bait was performed. One clone that was identified by 0 this procedure included amino acids encoded by nucleotides 1110-1750 of tankyrase (GB accession no. AF082557). EXAMPLE 69 Identification of IRAP/PTPZ Interaction 5 A yeast two-hybrid system as described in Example 1 using amino acids encoded by nucleotides 62-880 of IRAP (GB accession no. U62768) as bait was performed. One clone that was identified by this procedure included amino acids 1420-1780 of PTPZ (SP accession no. P23471). EXAMPLE 70 0 Identification of IRAP/3Spectrin Interaction A yeast two-hybrid system as described in Example 1 using amino acids encoded by nucleotides 2311-3136 of IRAP (GB accession no. U62768) as bait was performed. One clone that WO 00/65340 PCT/US00/10651 58 was identified by this procedure included amino acids 1487-1993 of P-spectrin (SP accession no. Q01082). EXAMPLE 71 5 Identification of IRAP/PI-3K85 Interaction A yeast two-hybrid system as described in Example 1 using amino acids encoded by nucleotides 2311-3136 of IRAP (GB accession no. U62768) as bait was performed. One clone that was identified by this procedure included amino acids 498-544 of PI-3 kinase p85 subunit (SP accession no. P27986). 0 EXAMPLE 72 Identification of PP5/HSP89 Interaction A yeast two-hybrid system as described in Example 1 using amino acids 1-150 of PP5 (SP accession no. P53041) as bait was performed. One clone that was identified by this procedure 5 included amino acids 517-681 of Hsp89 (SP accession no. P07900). EXAMPLE 73 Identification of PP5/Tankyrase Interaction A yeast two-hybrid system as described in Example 1 using amino acids 1-150 of PP5 (SP 0 accession no. P53041) as bait was performed. One clone that was identified by this procedure included amino acids encoded by nucleotides 1110-1750 and nucleotides 1809-2257 of tankyrase (GB accession no. AF082557). EXAMPLE 74 5 Identification of PI-3K85/Tankvrase Interaction A yeast two-hybrid system as described in Example 1 using amino acids 320-440 of PI-3 kinase p85 subunit (SP accession no. P27986) as bait was performed. Two clones that were identified by this procedure included amino acids encoded by nucleotides 1110-1750 of tankyrase (GB accession no. AF082557). 0 WO 00/65340 PCT/US00/10651 59 EXAMPLE 75 Identification of PI-3K110/APP Interaction A yeast two-hybrid system as described in Example 1 using amino acids 1-300 of PI-3 kinase p 1 10 subunit (SP accession no. P42338) as bait was performed. One clone that was 5 identified by this procedure included amino acids 374-546 ofAPP (SP accession no. P05067). EXAMPLE 76 Generation of Polyclonal Antibody Against Protein Complexes As shown above, Glut4 interacts with CARP to form a complex. A complex of the two 10 proteins is prepared, e.g., by mixing purified preparations of each of the two proteins. If desired, the protein complex can be stabilized by cross-linking the proteins in the complex, by methods known to those of skill in the art. The protein complex is used to immunize rabbits and mice using a procedure similar to that described by Harlow et al. (1988). This procedure has been shown to generate Abs against various other proteins (for example, see Kraemer et al., 1993). 5 Briefly, purified protein complex is used as immunogen in rabbits. Rabbits are immunized with 100 pg of the protein in complete Freund's adjuvant and boosted twice in three-week intervals, first with 100 pg of immunogen in incomplete Freund's adjuvant, and followed by 100 gg of immunogen in PBS. Antibody-containing serum is collected two weeks thereafter. The antisera is preadsorbed with Glut4 and CARP, such that the remaining antisera comprises antibodies which 20 bind conformational epitopes, i.e., complex-specific epitopes, present on the Glut4-CARP complex but not on the monomers. Polyclonal antibodies against each of the complexes set forth in Tables 1-73 are prepared in a similar manner by mixing the specified proteins together, immunizing an animal and isolating antibodies specific for the protein complex, but not for the individual proteins. 25 WO 00/65340 PCT/US00/10651 60 EXAMPLE 77 Generation of Monoclonal Antibodies Specific for Protein Complexes Monoclonal antibodies are generated according to the following protocol. Mice are immunized with immunogen comprising Glut4/CARP complexes conjugated to keyhole limpet 5 hemocyanin using glutaraldehyde or EDC as is well known in the art. The complexes can be prepared as described in Example 76, and may also be stabilized by cross-linking. The immunogen is mixed with an adjuvant. Each mouse receives four injections of 10 to 100 pg of immunogen, and after the fourth injection blood samples are taken from the mice to determine if the serum contains antibody to the immunogen. Serum titer is determined by ELISA or RIA. Mice with sera indicating 10 the presence of antibody to the immunogen are selected for hybridoma production. Spleens are removed from immune mice and a single-cell suspension is prepared (Harlow et al., 1988). Cell fusions are performed essentially as described by Kohler et al. (1975). Briefly, P3.65.3 myeloma cells (American Type Culture Collection, Rockville, MD) or NS-1 myeloma cells are fused with immune spleen cells using polyethylene glycol as described by Harlow et al. (1988). 15 Cells are plated at a density of 2x10 5 cells/well in 96-well tissue culture plates. Individual wells are examined for growth, and the supernatants of wells with growth are tested for the presence of Glut4/CARP complex-specific antibodies by ELISA or RIA using Glut4/CARP complex as target protein. Cells in positive wells are expanded and subcloned to establish and confirm monoclonality. Clones with the desired specificities are expanded and grown as ascites in mice or in a 20 hollow fiber system to produce sufficient quantities of antibodies for characterization and assay development. Antibodies are tested for binding to Glut4 alone or to CARP alone, to determine which are specific for the Glut4/CARP complex as opposed to those that bind to the individual proteins. Monoclonal antibodies against each of the complexes set forth in Tables 1-73 are prepared 25 in a similar manner by mixing the specified proteins together, immunizing an animal, fusing spleen cells with myeloma cells and isolating clones which produce antibodies specific for the protein complex, but not for the individual proteins. EXAMPLE 78 30 In vitro Identification of Modulators for Protein-Protein Interactions The present invention is useful in screening for agents that modulate the interaction of Glut4 and CARP. The knowledge that Glut4 and CARP form a complex is useful in designing such WO 00/65340 PCT/US00/10651 61 assays. Candidate agents are screened by mixing Glut4 and CARP (a) in the presence of a candidate agent, and (b) in the absence of the candidate agent. The amount of complex formed is measured for each sample. An agent modulates the interaction of Glut4 and CARP if the amount of complex formed in the presence of the agent is greater than (promoting the interaction), or less than 5 (inhibiting the interaction) the amount of complex formed in the absence of the agent. The amount of complex is measured by a binding assay, which shows the formation of the complex, or by using antibodies immunoreactive to the complex. Briefly, a binding assay is performed in which immobilized Glut4 is used to bind labeled CARP. The labeled CARP is contacted with the immobilized Glut4 under aqueous conditions that 0 permit specific binding of the two proteins to form an Glut4/CARP complex in the absence of an added test agent. Particular aqueous conditions may be selected according to conventional methods. Any reaction condition can be used as long as specific binding of Glut4/CARP occurs in the control reaction. A parallel binding assay is performed in which the test agent is added to the reaction mixture. The amount of labeled CARP bound to the immobilized Glut4 is determined for the 5 reactions in the absence or presence of the test agent. If the amount of bound, labeled CARP in the presence of the test agent is different than the amount of bound labeled CARP in the absence of the test agent, the test agent is a modulator of the interaction of Glut4 and CARP. Candidate agents for modulating the interaction of each of the protein complexes set forth in Tables 1-73 are screened in vitro in a similar manner. 0 EXAMPLE 79 In vivo Identification of Modulators for Protein-Protein Interactions In addition to the in vitro method described in Example 78, an in vivo assay can also be used to screen for agents which modulate the interaction of Glut4 and CARP. Briefly, a yeast two-hybrid 5 system is used in which the yeast cells express (1) a first fusion protein comprising Glut4 or a fragment thereof and a first transcriptional regulatory protein sequence, e.g., GAL4 activation domain, (2) a second fusion protein comprising CARP or a fragment thereof and a second transcriptional regulatory protein sequence, e.g., GAL4 DNA-binding domain, and (3) a reporter gene, e.g., -galactosidase, which is transcribed when an intermolecular complex comprising the 0 first fusion protein and the second fusion protein is formed. 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While the invention has been disclosed in this patent application by reference to the details of preferred embodiments of the invention, it is to be understood that the disclosure is intended in an illustrative rather than in a limiting sense, as it is contemplated that modifications will readily 0 occur to those skilled in the art, within the spirit of the invention and the scope of the appended claims. LIST OF REFERENCES Annaert, W.G. et al. (1997). JCell Biol. 139:1397-410. 5 Aronheim et al., 1997. Mol. Cell. Biol. 17:3094-3102. Bartel, P.L. et al. (1993). "Using the 2-hybrid system to detect protein-protein interactions." In: Cellular Interactions in Development: A Practical Approach, Oxford University Press, pp. 153-179. Bartel, P.L. et al. (1996). Nat Genet 12:72-77. 0 Bartel, P.L. and Fields, S. (1997). The Yeast Two-Hybrid System. New York: Oxford University Press. Ben-Ze'ev, A. et al. (1998). Curr Opin Cell Biol. 10:629-39. Burch, G.H. et al. (1997). Nat Genet. 17:104-8. 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WO 00/65340 PCT/US00/10651 65 Sukhatme, V.P. et al. (1988). Cell 53:37-43. Thorson JA, et al. (1998). Mol Cell Biol. 18:5229-38. Tisdale, E.J. et al. (1998). J Biol Chem. 273:17269-77. Uphues, I. et al. (1994). Biochem J. 301:177-82. 5 Vergani, D. et al. (1985). Lancet II:294-298. Vollenweider, P. et al. (1997). Endocrinology 138:4941-9. Wang, X.S. et al. (1998). Biochem Biophys Res Commun. 253:33-7. Wang, Z. et al. (1999). FEBSLett. 453:135-9. Waters, S.B. et al. (1997). JBiol Chem. 272:23323-7. 0 Weir, M.L. et al. (1998). Biochem J 333:247-51. Wigle, J.T. et al. (1997). JBiol Chem. 272:32384-94. Yaffe, M.B. et al. (1997). Cell 91:961-71. Zierath, J.R. et al. (1998). Mol Cell Biochem. 182:153-60. 5 PCT Published Application No. WO 97/27296 PCT Published Application No. WO 99/65939 U.S. Patent No. 5,622,852 U.S. Patent No. 5,773,218

Claims

1. An isolated protein complex comprising two proteins, the protein complex selected from the group consisting of 5 (a) a complex set forth in Table 1; (b) a complex set forth in Table 2; (c) a complex set forth in Table 3; (d) a complex set forth in Table 4; (e) a complex set forth in Table 5; 0 (f) a complex set forth in Table 6; (g) a complex set forth in Table 7; (h) a complex set forth in Table 8; (i) a complex set forth in Table 9; (j) a complex set forth in Table 10; 5 (k) a complex set forth in Table 11; (1) a complex set forth in Table 12; (m) a complex set forth in Table 13; (n) a complex set forth in Table 14;. (o) a complex set forth in Table 15; 0 (p) a complex set forth in Table 16; (q) a complex set forth in Table 17; (r) a complex set forth in Table 18; (s) a complex set forth in Table 19; (t) a complex set forth in Table 20; !5 (u) a complex set forth in Table 21; (v) a complex set forth in Table 22; (w) a complex set forth in Table 23; (x) a complex set forth in Table 24; (y) a complex set forth in Table 25; 0 (z) a complex set forth in Table 26; (aa) a complex set forth in Table 27; (ab) a complex set forth in Table 28; WO 00/65340 PCT/US00/10651 67 (ac) a complex set forth in Table 29; (ad) a complex set forth in Table 30; (ae) a complex set forth in Table 31; (af) a complex set forth in Table 32; 5 (ag) a complex set forth in Table 33; (ah) a complex set forth in Table 34; (ai) a complex set forth in Table 35; (aj) a complex set forth in Table 36; (ak) a complex set forth in Table 37; ) (al) a complex set forth in Table 38; (am) a complex set forth in Table 39; (an) a complex set forth in Table 40; (ao) a complex set forth in Table 41;. (ap) a complex set forth in Table 42; 5 (aq) a complex set forth in Table 43; (ar) a complex set forth in Table 44; (as) a complex set forth in Table 45 (at) a complex set forth in Table 46; (au) a complex set forth in Table 47; ) (av) a complex set forth in Table 48; (aw) a complex set forth in Table 49; (ax) a complex set forth in Table 50; (ay) a complex set forth in Table 51; (az) a complex set forth in Table 52; 5 (ba) a complex set forth in Table 53; (bb) a complex set forth in Table 54; (bc) a complex set forth in Table 55; (bd) a complex set forth in Table 56; (be) a complex set forth in Table 57; S(bf) a complex set forth in Table 58; (bg) a complex set forth in Table 59; (bh) a complex set forth in Table 60; WO 00/65340 PCT/US00/10651 68 (bi) a complex set forth in Table 61; (bj) a complex set forth in Table 62; (bk) a complex set forth in Table 63; (bl) a complex set forth in Table 64; (bm) a complex set forth in Table 65; (bn) a complex set forth in Table 66; (bo) a complex set forth in Table 67; (bp) a complex set forth in Table 68;. (bq) a complex set forth in Table 69; D (br) a complex set forth in Table 70; (bs) a complex set forth in Table 71; (bt) a complex set forth in Table 72; and (bu) a complex set forth in Table 73. 5

2. The protein complex of claim 1, wherein said protein complex comprises complete proteins.

3. The protein complex of claim 1, wherein said protein complex comprises a fragment of one protein and a complete protein of anther protein. 0

4. The protein complex of claim 1, wherein said protein complex comprises fragments of proteins.

5. An isolated antibody selectively immunoreactive with the protein complex of claim 1. .5

6. The antibody of claim 5, wherein said antibody is a monoclonal antibody.

7. A method for diagnosing a physiological disorder in an animal, which comprises assaying for: (a) whether a protein complex set forth in any one of Tables 1-73 is present in a 30 tissue extract; (b) the ability of proteins to form a protein complex set forth in any one of Tables 1-73; and WO 00/65340 PCT/US00/10651 69 (c) a mutation in a gene encoding a protein of a protein complex set forth in any one of Tables 1-73.

8. The method of claim 7, wherein said animal is a human. 5

9. The method of claim 7, wherein the diagnosis is for a predisposition to said physiological disorder.

10. The method of claim 7, wherein the diagnosis is for the existence of said physiological 0 disorder.

11. The method of claim 7, wherein said assay comprises a yeast two-hybrid assay.

12. The method of claim 7, wherein said assay comprises measuring in vitro a complex formed 5 by combining the proteins of the protein complex, said proteins isolated from said animal.

13. The method of claim 12, wherein said complex is measured by binding with an antibody specific for said complex. 20

14. The method of claim 7, wherein said assay comprises mixing an antibody specific for said protein complex with a tissue extract from said animal and measuring the binding of said antibody.

15. A method for determining whether a mutation in a gene encoding one of the proteins of a 25 protein complex set forth in any one of Tables 1-73 is useful for diagnosing a physiological disorder, which comprises assaying for the ability of said protein with said mutation to form a complex with the other protein of said protein complex, wherein an inability to form said complex is indicative of said mutation being useful for diagnosing a physiological disorder. 30

16. The method of claim 15, wherein said gene is an animal gene.

17. The method of claim 16, wherein said animal is a human. WO 00/65340 PCT/US00/10651 70

18. The method of claim 15, wherein the diagnosis is for a predisposition to a physiological disorder. 5

19. The method of claim 15, wherein the diagnosis is for the existence of a physiological disorder.

20. The method of claim 15, wherein said assay comprises a yeast two-hybrid assay. 0

21. The method of claim 15, wherein said assay comprises measuring in vitro a complex formed by combining the proteins of the protein complex, said proteins isolated from an animal.

22. The method of claim 21, wherein said animal is a human. 5

23. The method of claim 21, wherein said complex is measured by binding with an antibody specific for said complex.

24. A method for screening for drug candidates capable of modulating the interaction of the proteins of a protein complex set forth in any one of Tables 1-73, which comprises: 0 (a) combining the proteins of said protein complex in the presence of a drug to form a first complex; (b) combining the proteins in the absence of said drug to form a second complex; (c) measuring the amount of said first complex and said second complex; and (d) comparing the amount of said first complex with the amount of said second 5 complex, wherein if the amount of said first complex is greater than, or less than the amount of said second complex, then the drug is a drug candidate for modulating the interaction of the proteins of said protein complex.. 0

25. The method of claim 24, wherein said screening is an in vitro screening. WO 00/65340 PCT/US00/10651 71

26. The method of claim 24, wherein said complex is measured by binding with an antibody specific for said protein complexes.

27. The method of claim 24, wherein if the amount of said first complex is greater than the 5 amount of said second complex, then said drug is a drug candidate for promoting the interaction of said proteins.

28. The method of claim 24, wherein if the amount of said first complex is less than the amount of said second complex, then said drug is a drug candidate for inhibiting the interaction of 0 said proteins.

29. A non-human animal model for a physiological disorder wherein the genome of said animal or an ancestor thereof has been modified such that the formation of a protein complex set forth in any one of Tables 1-73 has been altered. 5

30. The non-human animal model of claim 29, wherein the formation of said protein complex has been altered as a result of: (a) over-expression of at least one of the proteins of said protein complex; (b) replacement of a gene for at least one of the proteins of said protein complex with 0 a gene from a second animal and expression of said protein; (c) expression of a mutant form of at least one of the proteins of said protein complex; (d) a lack of expression of at least one of the proteins of said protein complex; or (e) reduced expression of at least one of the proteins of said protein complex. 25

31. A cell line obtained from the animal model of claim 29.

32. A non-human animal model for a physiological disorder, wherein the biological activity of a protein complex set forth in any one of Tables 1-73 has been altered. 30

33. The non-human animal model of claim 32, wherein said biological activity has been altered as a result of: WO 00/65340 PCT/US00/10651 72 (a) disrupting the formation of said complex; or (b) disrupting the action of said complex.

34. The non-human animal model of claim 32, wherein the formation of said complex is 5 disrupted by binding an antibody to at least one of the proteins which form said protein complex.

35. The non-human animal model of claim 32, wherein the action of said complex is disrupted by binding an antibody to said complex. 0

36. The non-human animal model of claim 32, wherein the formation of said complex is disrupted by binding a small molecule to at least one of the proteins which form said protein complex. 15

37. The non-human animal model of claim 32, wherein the action of said complex is disrupted by binding a small molecule to said complex.

38. A cell in which the genome of cells of said cell line has been modified to produce at least one protein complex set forth in any one of Tables 1-73. 20

39. A cell line in which the genome of the cells of said cell line has been modified to eliminate at least one protein of a protein complex set forth in any one of Tables 1-73.

40. A method of screening for drug candidates useful in treating a physiological disorder 25 which comprises the steps of: (a) measuring the activity of a protein selected from the proteins set forth in Tables 1-73 in the presence of a drug, (b) measuring the activity of said protein in the absence of said drug, and (c) comparing the activity measured in steps (1) and (2), 30 wherein if there is a difference in activity, then said drug is a drug candidate for treating said physiological disorder. WO 00/65340 PCT/US00/10651 73

41. An isolated nucleic acid encoding a polypeptide comprising an amino acid sequence selected from the group consisting of amino acid sequences set forth in SEQ ID NOs:4, 6, 8 andl0.

42. The isolated nucleic acid of claim 41 comprising a nucleotide sequence selected from the 5 group consisting of nucleotide sequences set forth in SEQ ID NOs:3, 5, 7 and 9.

43. An isolated polypeptide comprising an amino acid amino acid sequence selected from the group consisting of amino acid sequences set forth in SEQ ID NOs:4, 6, 8 and10. 0