The heterotrimeric guanine nucleotide binding protein (G-protein) coupled receptors (GPCR) contain seven hydrophobic membrane-spanning regions. Thus, their intracellular domains consist of three loops and one carboxyl terminus. It is known that the intracellular domains, especially the short fragments near the amino- and carboxyl termini of the third intracellular loop (i3 loop) are critical for interaction with and activate of G-proteins. Recent evidence indicates that some proteins other than G-proteins, so called receptor interaction partners (RIPs), can interact with the carboxyl termini and the i3 loops of some GPCRs. Although only a minute percentage of the RIPs for some GPCRs have been examined in detail, careful studies might yield important clues to the roles of intracellular domains of GPCRs. The long-term objective of my laboratory is to identify novel signaling mechanisms of the angiotensin II subtype 1 receptor (AT1R), and eventually to develop novel therapeutics that target the interface between the receptor and its interacting proteins. The short-term objective of this K award proposal is to define novel signaling protein complexes that are associated with the AT1R intracellular domains, and to explore their functional significance. The initial work will focus on the identification of novel RIPs in rat aortic vascular smooth muscle cells (RASM), which interact with the carboxyl terminus (AT1aR-CT) and/or i3 loop (AT1aR-i3L) of the AT1aR. Our strategy will use glutathione S-transferase (GST) fusion proteins, "pull-down" assays and mass spectrometry. We will approach our short-term goals with two specific aims.1. To identify novel receptor interaction partners (RIPs) in RASM, which interact with AT1aR-CT and/or AT1aR-i3L.2. To identify the domains of AT1aR-CT and/or AT1aR-i3L that interact with the novel RIPsThis project will provide me with the opportunity to blend my previous experience in studying GPCR protein-protein interactions with new opportunities to use mass spectrometric methods to study GPCRs. Because of the emerging importance of the proteome, successful completion of this project will provide me with a solid foundation for developing an independent and self-sustaining research program.