Dynamin and β-Arrestin Reveal Distinct Mechanisms for G Protein-coupled Receptor Internalization

Abstract

The process of agonist-promoted internalization (sequestration) of G protein-coupled receptors (GPCRs) is intimately linked to the regulation of GPCR responsiveness. Following agonist-mediated desensitization, sequestration of GPCR is presumably associated with the dephosphorylation and recycling of functional receptors. However, the exact mechanisms responsible for GPCR sequestration, even for the prototypic ␤ 2 -adrenergic receptor (␤ 2 AR), have remained controversial. We demonstrate here that dynamin, a GTPase that regulates the formation and internalization of clathrincoated vesicles, is essential for the agonist-promoted sequestration of the ␤ 2 AR, suggesting that the ␤ 2 AR internalizes via the clathrin-coated vesicle-mediated endocytic pathway. In contrast, internalization of the angiotensin II type 1A receptor (AT 1A R), another typical GPCR, does not require dynamin. In addition, the AT 1A R internalizes independent of the function of ␤-arrestin, a critical component for ␤ 2 AR cellular trafficking, but additional AT 1A Rs are mobilized to the dynamin-dependent pathway upon overexpression of ␤-arrestin. These findings demonstrate that GPCRs can utilize distinct endocytic pathways, distinguishable by dynamin and ␤-arrestin, and that ␤-arrestins function as adaptor proteins specifically targeting GPCRs for dynamin-dependent endocytosis via clathrin-coated vesicles. G protein-coupled receptor (GPCR)1 internalization is a phenomenon triggered by agonist stimulation. This rapid internalization, referred to as sequestration (1), follows the agonistmediated phosphorylation and desensitization of the signaling function of these receptors and is thought to contribute to the resensitization of GPCR responsiveness (2-5). Although the process of sequestration has been documented for many GPCRs, the cellular nature and molecular determinants of sequestration have remained elusive. Recently, however, phosphorylation of GPCRs by specific G protein-coupled receptor kinases has been demonstrated to facilitate agonist-mediated receptor sequestration (6, 7). This receptor phosphorylation serves to increase the affinity of GPCRs for ␤-arrestin proteins, which, in addition to uncoupling receptor-G protein interactions, can act as adaptor-like molecules for receptor trafficking (8).Considerable evidence exists that GPCRs internalize via the clathrin-coated vesicle-mediated endocytic pathway (9 -12). However, even for the prototypic GPCR, ␤ 2 -adrenergic receptor (␤ 2 AR), this issue has remained controversial (9, 10, 13). Dynamin, a 100-kDa GTPase, originally isolated as a nucleotidedependent microtubule binding protein, has been identified as a major component and marker of the clathrin-mediated endocytic pathway (14 -18). Dynamin colocalizes with clathrin (19,20) and binds to the appendage domain of ␣-adaptin, a component of the clathrin-coated pits (21). Functionally, dynamin contributes to the early stages of endocytosis by catalyzing a GTP-dependent pinching off of endocytic vesicles from the plasma membrane (22,23). ...

Regulation of Human Chemokine Receptors CXCR4

“…Internalization of type A cholecystokinin receptor was unaffected by C-terminal truncation, whereas internalization of type B cholecystokinin receptor was significantly reduced in a C-terminal truncation mutant (49). While arrestins and dynamin appear to play an important role in phosphorylation-dependent sequestration of G-protein-coupled receptors (43,46), the molecular mechanisms of phosphorylation-independent sequestration observed here with CXCR4 and previously with other G-protein-coupled receptors (49) remain to be determined.…”

“…Multiple mechanisms for internalization of surface proteins were described (40,46,47). Recent studies have demonstrated that phosphorylation at either one of the two independent clusters of phosphorylation sites in m2-muscarinic receptors is sufficient for agonist-induced internalization of the receptor, whereas mutation of both clusters severely impaired internalization (48).…”

Differential β-arrestin2 requirements for constitutive and agonist-induced internalization of the CB1 cannabinoid receptor

“…This is important because various examples demonstrate that β-arrestin2 recruitment and receptor internalization can be independent processes, as it is the case e.g. with the PAR1 thrombin receptor (Paing et al, 2002), the CXCR2 chemokine receptor (Fan et al, 2001;Zhao et al, 2004), the N-formyl peptide receptor (FPR) (Vines et al, 2003), or at high agonist concentration the AT 1 angiotensin receptor (Gaborik et al, 2001;Hunyady and Catt, 2006;Zhang et al, 1996). Therefore, it is important to study directly the β-arrestin dependence of CB 1 R internalization.…”

Regulatory mechanisms that modulate signalling by G-protein-coupled receptors

“…Thus arrestins may function as adaptor molecules that may recruit cellular proteins that facilitate receptor endocytosis, or may directly mediate endocytosis themselves. Dynamin may be one protein that interacts with arrestins to enhance endocytosis of some receptors [180]. Dynamin is important for internalization of the β # -AR, since overexpression of a dynamin GTPase mutant inhibits agonist-induced endocytosis.…”