SAP97 controls the trafficking and resensitization of the beta-1-adrenergic receptor through its PDZ2 and I3 domains

PLoS One. 2013 May 16;8(5):e63379. doi: 10.1371/journal.pone.0063379. Print 2013.

Abstract

Previous studies have determined that the type-1 PDZ sequence at the extreme carboxy-terminus of the ß1-adrenergic receptor (ß1-AR) binds SAP97 and AKAP79 to organize a scaffold involved in trafficking of the ß1-AR. In this study we focused on characterizing the domains in SAP97 that were involved in recycling and resensitization of the ß1-AR in HEK-293 cells. Using a SAP97 knockdown and rescue strategy, we determined that PDZ-deletion mutants of SAP97 containing PDZ2 rescued the recycling and resensitization of the ß1-AR. Among the three PDZs of SAP97, PDZ2 displayed the highest affinity in binding to the ß1-AR. Expression of isolated PDZ2, but not the other PDZs, inhibited the recycling of the ß1-AR by destabilizing the macromolecular complex involved in trafficking and functional resensitization of the ß1-AR. In addition to its PDZs, SAP97 contains other protein interacting domains, such as the I3 sequence in the SRC homology-3 (SH3) domain, which binds to AKAP79. Deletion of I3 from SAP97 (ΔI3-SAP97) did not affect the binding of SAP97 to the ß1-AR. However, ΔI3-SAP97 could not rescue the recycling of the ß1-AR because it failed to incorporate AKAP79/PKA into the SAP97-ß1-AR complex. Therefore, bipartite binding of SAP97 to the ß1-AR and to AKAP79 is necessary for SAP97-mediated effects on recycling, externalization and functional resensitization of the ß1-AR. These data establish a prominent role for PDZ2 and I3 domains of SAP97 in organizing the ß1-adrenergic receptosome involved in connecting the ß1-AR to trafficking and signaling networks.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / chemistry
  • Adaptor Proteins, Signal Transducing / genetics
  • Adaptor Proteins, Signal Transducing / metabolism*
  • Animals
  • Cell Line
  • Humans
  • Immunoprecipitation
  • Membrane Proteins / chemistry
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Protein Binding
  • Protein Structure, Tertiary
  • Protein Transport
  • Rats
  • Receptors, Adrenergic, beta-1 / genetics
  • Receptors, Adrenergic, beta-1 / metabolism*
  • Signal Transduction

Substances

  • Adaptor Proteins, Signal Transducing
  • Dlg1 protein, rat
  • Membrane Proteins
  • Receptors, Adrenergic, beta-1