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PNAS 96 (22): 12559-12564

Copyright © 1999 by the National Academy of Sciences.


Identification of the endophilins (SH3p4/p8/p13) as novel binding partners for the β1-adrenergic receptor

Yuting Tang*,{dagger}, Liaoyuan A. Hu*,{dagger}, William E. Miller*,{dagger}, Niels Ringstad{ddagger}, Randy A. Hall{dagger},§, Julie A. Pitcher{dagger}, Pietro DeCamilli{ddagger}, and Robert J. Lefkowitz*,{dagger},||

*Howard Hughes Medical Institute and {dagger}Department of Medicine (Cardiology) and Biochemistry, P.O. Box 3821, Duke University Medical Center, Durham, NC 27710; and {ddagger}Yale University School of Medicine, New Haven, CT 06150

Contributed by Robert J. Lefkowitz

Accepted for publication September 1, 1999.

Abstract: Several G-protein coupled receptors, such as the β1-adrenergic receptor (β1-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 β1-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 β1-AR. In vitro and in human embryonic kidney (HEK) 293 cells, SH3p4 specifically binds to the third intracellular loop of the β1-AR but not to that of the β2-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 β1-ARs in HEK293 cells while having no effect on β2-ARs. Thus, our studies demonstrate a role of the SH3p4/p8/p13 protein family in β1-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.

§ Present address: Department of Pharmacology, Rollins Research Center, Emory University School of Medicine, Atlanta, GA 30322.

Present address: Medical Research Council Laboratory for Molecular Cell Biology, University College London, Gower Street, London, United Kingdom, WC1E 6BT.

|| To whom reprint requests should be addressed. E-mail: lefko001{at}

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