Identification of the endophilins (SH3p4/p8/p13) as novel binding partners for the beta1-adrenergic receptor

Proc Natl Acad Sci U S A. 1999 Oct 26;96(22):12559-64. doi: 10.1073/pnas.96.22.12559.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adaptor Proteins, Signal Transducing*
  • Adrenergic beta-1 Receptor Agonists
  • Animals
  • Carrier Proteins / metabolism*
  • Cattle
  • Cell Line
  • GTP-Binding Protein alpha Subunits, Gs / metabolism
  • Humans
  • Proline / metabolism
  • Protein Binding
  • Receptors, Adrenergic, beta-1 / chemistry
  • Receptors, Adrenergic, beta-1 / metabolism*
  • src Homology Domains

Substances

  • Adaptor Proteins, Signal Transducing
  • Adrenergic beta-1 Receptor Agonists
  • Carrier Proteins
  • Receptors, Adrenergic, beta-1
  • SH3GL2 protein, human
  • Proline
  • GTP-Binding Protein alpha Subunits, Gs