UCSF

G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane signaling receptors known and account for roughly half of all pharmaceutical targets. Receptor regulation is known to influence cellular signaling strength and specificity, but the mechanisms conferring cellular-level regulation are largely unclear. An important process impacting cellular sensitivity is the post-endocytic sorting of receptors between a downregulating lysosomal fate and a resensitizing recycling pathway to the cell surface. GPCR recycling, in particular, is itself a regulated process that requires recognition of cytoplasmic receptor motifs. The first motif recognized as a recycling sequence is a protein interaction ligand found at the C-terminus of the β2 adrenoceptor. This ligand has shown affinity for multiple proteins, several of which have been proposed to mediate recycling of the receptor. Using a combination of mutational, protein engineering, and RNA interference techniques to probe the recycling activity of three candidate interaction types in the HEK293 cell culture model, we have distinguished these interactions as necessary, sufficient, or dispensable with respect to mediating receptor recycling. In particular, we have identified a dichotomy between the sufficiency of the connectivity in the NHERF/ERM/Actin protein complex to promote recycling of engineered receptors and the necessity of a novel and functionally distinct interaction with Sortin Nexin 27 that is the only interaction detectably required for recycling of the β2 adrenoceptor. In addition, a competing interaction with the N-Ethylmaleimide Sensitive Factor was found to be relatively dispensable in this regard despite showing the capacity for physiologic fine-tuning of receptor trafficking and signaling. These results identify a new function of sorting nexin 27 in promoting PDZ-dependent recycling of an integral membrane protein, expand our basic understanding of trafficking mechanisms that regulate a prototypic signaling receptor, while additionally providing information critical to understanding the physiologic consequences of receptor regulation.