Identification of the endophilins (SH3p4/p8/p13) as novel binding partnersfor the beta 1-adrenergic receptor

Several C-protein coupled receptors, such as the beta 1-adrenergic receptor (beta 1-AR), contain polyproline motifs within their intracellular domains . Such motifs in other proteins are known to mediate protein-protein intera ctions such as with Src homology (SH)3 domains. Accordingly, we used the pr oline-rich third intracellular loop of the beta 1-AR either as a glutathion e S-transferase fusion protein in biochemical "pull-down" assays or as bait in the yeast two-hybrid system to search for interacting proteins. Both ap proaches identified SH3p4/p8/p13 (also referred to as endophilin 1/2/3), a SH3 domain-containing protein family, as binding partners for the beta 1-AR . In vitro and in human embryonic kidney (HEK) 293 cells, SH3p4 specificall y binds to the third intracellular loop of the beta 1-AR but not to that of the beta 2-AR, Moreover, this interaction is mediated by the C-terminal SH 3 domain of SH3p4. Functionally, overexpression of SH3p4 promotes agonist-i nduced internalization and modestly decreases the Cs coupling efficacy of b eta 1-ARs in HEK293 cells while having no effect on beta 2-ARs. Thus, our s tudies demonstrate a role of the SH3p4/p8/p13 protein family in Pi-AR signa ling and suggest that interaction between proline-rich motifs and SH3-conta ining proteins may represent a previously underappreciated aspect of C-prot ein coupled receptor signaling.