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G protein-coupled receptor (GPCR) sequestration to endosomes is proposed to be the mechanism by which G protein-coupled receptor kinase (GRK)-phosphorylated receptors are dephosphorylated and resensitized. The identification of beta-arrestins as GPCR trafficking molecules suggested that beta-arrestins might represent critical determinants for GPCR resensitization. Therefore, we tested whether beta2-adrenergic receptor (beta2AR) resensitization was dependent upon beta-arrestins and an intact clathrin-coated vesicle endocytic pathway. The overexpression of either the beta-arrestin 1-V53D dominant negative inhibitor of beta2AR sequestration or dynamin I-K44A to block clathrin-coated vesicle-mediated endocytosis impaired both beta2AR dephosphorylation and resensitization. In contrast, resensitization of a sequestration-impaired beta2AR mutant (Y326A) was reestablished following the overexpression of either GRK2 or beta-arrestin 1. Moreover, beta2ARs did not resensitize in COS-7 cells as the consequence of impaired sequestration and dephosphorylation. However, beta2AR resensitization was restored in these cells following the overexpression of beta-arrestin 2. These findings demonstrate, using both loss and gain of function paradigms, that beta2AR dephosphorylation and resensitization are dependent upon an intact sequestration pathway. These studies also indicate that beta-arrestins play an integral role in regulating not only the desensitization and intracellular trafficking of GPCRs but their ability to resensitize. beta-Arrestin expression levels appear to underlie cell type-specific differences in the regulation of GPCR resensitization.
Zhang et al. (Wed,) studied this question.