We plan to extend our previous studies on mechanisms underlying regulation of OigARs, by investigating the scaffolding protein spinophilin in aiaAR signaling/ trafficking. Preliminary data demonstrate spinophilin interacts with aiaARs witfi higher affinity than ait>ARs We also plan to extend our studies by studying matnx metalloproteases (MMPs) in mediating aiaAR signaling via EGFR transactivation compared with aibARs. Here we have a powerful new tool, a.naturally occurnng human 3"* intracellular loop aia-SNP G247R, which in our preliminary data provides differential activation of specific MMP subtypes Finally, while aims 1 &2 explore targeted, clinically relevant signaling proteins/ pathways, we recognize the importance of exploring novel binding partners Therefore our last specific aim will use Tandem-Affinity Purification combined with mass spectrometry to identify novel multi-protein complexes (the broader OiaAR signalosome) important in OiaAR signaling/ trafficking.- Unique resources and collaborators available at the Univ of WA make this approach both feasible and quantitative. AIM 1: Targeted examination ofthe scaffold protein spinophilin in aiaAR signaling and trafficking Hypothesis. Spinophilin binding to the OigAR 3^ loop mediates distinct subtype signalingArafficking 1 a Characterize spinophilin/oiaAR 3^*^ loop binding (co-immunoprecipitation assays, structure/function - analysisryeast 2-hybrid effects) &altered signaling pathways-(specific inhibitors,-siRNAs) [unreadable] -[unreadable] 1 b Examine role of spinophilin in OiaAR constitutive &agonist-induced trafficking , 1 c. Compare OiaAR sphinophilin-modulated signaling effects with OibAR and OiaAR 3"* loop SNP G247R AIIM 2: Investigate role of IMMPs in EGFR transactivation in aiaAR signaling Hypothesis: Differential activation of specific MMPs by aiAR subtypes mediates distinct signalingArafTicking 2 a Characterize role of MMPs in EGFR transactivation for differential OiaAR signaling (WT vs G247SNP) 2 b Examine gene expression arrays for OigAR WT ve'rsus G247 SNP &aib to identify specific MMPs involved (confirm using specific transactivation assays and inhibitors) AIM 3'Utilize genomic/proteomic approaches to identify broader aiaAR signalosome Hypothesis. Proteomics can be used to identify novel proteins in broader multi-protein complexes (signalosome) important in unique aigAR signaling and cellular trafficking 3.a Create tandem-affinity purification (TAP)-tag constructs for OigAR, OibAR Oia-SNP, establish cell lines 3.b Use proteomics-tandem reverse-phase liquid chromatography mass spectrometry (LC-MS/MS, LTQ- FT) technology to identify multi-protein complexes important in aia versus OibAR signaling/trafficking 3 c Perturb system (agonist/antagonist drugs, G247R Oi^-SNP) to identify differential signalosomes' 3 d Compare OiaAR signalosomes in 4 cell lines commonly used in OigAR studies (rat/human with different . surface versus intracellular receptor expression to identify key proteins modulating OiaAR trafficking RELEVANCE (See instructions) ' QiaARs are important in many diseases such as hypertension, blood vessel repair, prostate and myocardial hypertrophy, and myocardial ischemia Understanding how these receptors are regulated, particularly in their unique cellular environments, provides new understanding and drug targets for disease treatment