A central role for beta-arrestins and clathrin-coated vesicle-mediated endocytosis in beta(2)-adrenergic receptor resensitization - Differential regulation of receptor resensitization in two distinct cell types

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 beta(2)-adrenergic receptor (beta(2)AR) 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 beta(2)AR sequestration or dynamin I-K44A to block, clathrin-coated vesicle-mediated endocytosis impaired both beta(2)AR dephosphorylation and resensitization. In contrast, resensitization of a sequestration impaired beta(2)AR mutant (Y326A) was reestablished following the overexpression of either GRK2 or beta-arrestin 1. Moreover, beta(2)ARs did not resensitize in COS-7 cells as the consequence of impaired sequestration and dephosphorylation. However, beta(2)AR 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 beta(2)AR 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.