Identification of the endophilins (SH3p4/p8/p13) as novel binding partners for the beta1-adrenergic receptor.
Several G-protein coupled receptors, such as the beta1-adrenergic receptor (beta1-AR), contain polyproline motifs within their intracellular domains. Such motifs in other proteins are known to mediate protein-protein interactions such as with Src homology (SH)3 domains. Accordingly, we used the proline-rich third intracellular loop of the beta1-AR either as a glutathione S-transferase fusion protein in biochemical "pull-down" assays or as bait in the yeast two-hybrid system to search for interacting proteins. Both approaches identified SH3p4/p8/p13 (also referred to as endophilin 1/2/3), a SH3 domain-containing protein family, as binding partners for the beta1-AR. In vitro and in human embryonic kidney (HEK) 293 cells, SH3p4 specifically binds to the third intracellular loop of the beta1-AR but not to that of the beta2-AR. Moreover, this interaction is mediated by the C-terminal SH3 domain of SH3p4. Functionally, overexpression of SH3p4 promotes agonist-induced internalization and modestly decreases the Gs coupling efficacy of beta1-ARs in HEK293 cells while having no effect on beta2-ARs. Thus, our studies demonstrate a role of the SH3p4/p8/p13 protein family in beta1-AR signaling and suggest that interaction between proline-rich motifs and SH3-containing proteins may represent a previously underappreciated aspect of G-protein coupled receptor signaling.
Tang
Y
Y
Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC 27710, USA.
Hu
L A
LA
Miller
W E
WE
Ringstad
N
N
Hall
R A
RA
Pitcher
J A
JA
DeCamilli
P
P
Lefkowitz
R J
RJ
D048868:Adaptor Proteins, Signal Transducing; D058665:Adrenergic beta-1 Receptor Agonists; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism; D006801:Humans; D011392:Proline / Q000378:metabolism; D011485:Protein Binding; D018342:Receptors, Adrenergic, beta-1 / Q000737:chemistry / Q000378:metabolism*; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D058665:Adrenergic beta-1 Receptor Agonists; D002352:Carrier Proteins; D018342:Receptors, Adrenergic, beta-1; C107640:SH3GL2 protein, human; D011392:Proline; D019205:GTP-Binding Protein alpha Subunits, Gs