SAP97 Controls the Trafficking and Resensitization of the Beta-1-Adrenergic Receptor through Its PDZ2 and I3 Domains

Previous studies have determined that the type-1 PDZ sequence at the extreme carboxy-terminus of the ß(1)-adrenergic receptor (ß(1)-AR) binds SAP97 and AKAP79 to organize a scaffold involved in trafficking of the ß(1)-AR. In this study we focused on characterizing the domains in SAP97 that were involved in recycling and resensitization of the ß(1)-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 ß(1)-AR. Among the three PDZs of SAP97, PDZ2 displayed the highest affinity in binding to the ß(1)-AR. Expression of isolated PDZ2, but not the other PDZs, inhibited the recycling of the ß(1)-AR by destabilizing the macromolecular complex involved in trafficking and functional resensitization of the ß(1)-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 ß(1)-AR. However, ΔI3-SAP97 could not rescue the recycling of the ß(1)-AR because it failed to incorporate AKAP79/PKA into the SAP97-ß(1)-AR complex. Therefore, bipartite binding of SAP97 to the ß(1)-AR and to AKAP79 is necessary for SAP97-mediated effects on recycling, externalization and functional resensitization of the ß(1)-AR. These data establish a prominent role for PDZ2 and I3 domains of SAP97 in organizing the ß(1)-adrenergic receptosome involved in connecting the ß(1)-AR to trafficking and signaling networks.