Stimulation of beta2-adrenergic receptors on the cell surface by adrenaline or noradrenaline leads to alterations in the metabolism, excitability, differentiation and growth of many cell types. These effects have traditionally been thought to be mediated exclusively by receptor activation of intracellular G proteins. However, certain physiological effects of beta2-adrenergic receptor stimulation, notably the regulation of cellular pH by modulation of Na+/H+ exchanger (NHE) function, do not seem to be entirely dependent on G-protein activation. We report here a direct agonist-promoted association of the beta2-adrenergic receptor with the Na+/H+ exchanger regulatory factor (NHERF), a protein that regulates the activity of the Na+/H+ exchanger type 3 (NHE3). NHERF binds to the beta2-adrenergic receptor by means of a PDZ-domain-mediated interaction with the last few residues of the carboxy-terminal cytoplasmic domain of the receptor. Mutation of the final residue of the beta2-adrenergic receptor from leucine to alanine abolishes the receptor's interaction with NHERF and also markedly alters beta2-adrenergic receptor regulation of NHE3 in cells without altering receptor-mediated activation of adenylyl cyclase. Our findings indicate that agonist-dependent beta2-adrenergic receptor binding of NHERF plays a role in beta2-adrenergic receptor-mediated regulation of Na+/H+ exchange.

No abstract

G protein-coupled receptor activation leads to the membrane recruitment and activation of G proteincoupled receptor kinases, which phosphorylate receptors and lead to their inactivation. We have identified a novel G protein-coupled receptor kinase-interacting protein, GIT1, that is a GTPase-activating protein (GAP) for the ADP ribosylation factor (ARF) family of small GTP-binding proteins. Overexpression of GIT1 leads to reduced ␤ 2 -adrenergic receptor signaling and increased receptor phosphorylation, which result from reduced receptor internalization and resensitization. These cellular effects of GIT1 require its intact ARF GAP activity and do not ref lect regulation of GRK kinase activity. These results suggest an essential role for ARF proteins in regulating ␤ 2 -adrenergic receptor endocytosis. Moreover, they provide a mechanism for integration of receptor activation and endocytosis through regulation of ARF protein activation by GRK-mediated recruitment of the GIT1 ARF GAP to the plasma membrane.

␤-Arrestins are multifunctional adaptor proteins known to regulate internalization of agonist-stimulated G protein-coupled receptors by linking them to endocytic proteins such as clathrin and AP-2. Here we describe a previously unappreciated mechanism by which ␤-arrestin orchestrates the process of receptor endocytosis through the activation of ADP-ribosylation factor 6 (ARF6), a small GTP-binding protein. Involvement of ARF6 in the endocytic process is demonstrated by the ability of GTP-binding defective and GTP hydrolysisdeficient mutants to inhibit internalization of the ␤ 2 -adrenergic receptor. The importance of regulation of ARF6 function is shown by the ability of the ARF GTPase-activating protein GIT1 to inhibit and of the ARF nucleotide exchange factor, ARNO, to enhance receptor endocytosis. Endogenous ␤-arrestin is found in complex with ARNO. Upon agonist stimulation of the receptor, ␤-arrestin also interacts with the GDP-liganded form of ARF6, thereby facilitating ARNO-promoted GTP loading and activation of the G protein. Thus, the agonist-driven formation of a complex including ␤-arrestin, ARNO, and ARF6 provides a molecular mechanism that explains how the agonist-stimulated receptor recruits a small G protein necessary for the endocytic process and controls its activation.

The angiotensin type 1 receptor (AT1R) and its octapeptide ligand, angiotensin II (AngII), engage multiple downstream signaling pathways, including those mediated by heterotrimeric guanosine triphosphate-binding proteins (G proteins) and those mediated by β-arrestin. Here, we examined AT1R-mediated Gα(q) and β-arrestin signaling with multiple AngII analogs bearing substitutions at position 8, which is critical for binding to the AT1R and its activation of G proteins. Using assays that discriminated between ligand-promoted recruitment of β-arrestin to the AT1R and its resulting conformational rearrangement, we extend the concept of biased signaling to include the analog's propensity to differentially promote conformational changes in β-arrestin, two responses that were differentially affected by distinct G protein-coupled receptor kinases. The efficacy of AngII analogs in activating extracellular signal-regulated kinases 1 and 2 correlated with the stability of the complexes between β-arrestin and AT1R in endosomes, rather than with the extent of β-arrestin recruitment to the receptor. In vascular smooth muscle cells, the ligand-induced conformational changes in β-arrestin correlated with whether the ligand promoted β-arrestin-dependent migration or proliferation. Our data indicate that biased signaling not only occurs between G protein- and β-arrestin-mediated pathways but also occurred at the level of the AT1R and β-arrestin, such that different AngII analogs selectively engaged distinct β-arrestin conformations, which led to specific signaling events and cell responses.

Following agonist stimulation, most G protein-coupled receptors become desensitized and are internalized, either to be degraded or recycled back to the cell surface. What determines the fate of a specific receptor type after it is internalized is poorly understood. Here we show that the rapidly recycling ␤2 adrenergic receptor (␤2AR) binds via a determinant including the last three amino acids in its carboxyl-terminal tail to the membrane fusion regulatory protein, N-ethylmaleimidesensitive factor (NSF). This is documented by in vitro overlay assays and by cellular coimmunoprecipitations. Receptors bearing mutations in any of the last three residues fail to interact with NSF. After stimulation with the agonist isoproterenol, a green fluorescent protein fusion of NSF colocalizes with the wild type ␤2AR but not with a tail-mutated ␤2AR. The ␤2AR-NSF interaction is required for efficient internalization of the receptors and for their recycling to the cell surface. Mutations in the ␤2AR tail that ablate NSF binding reduce the efficiency of receptor internalization upon agonist stimulation. Upon subsequent treatment of cells with the antagonist propranolol, wild type receptors return to the cell surface, while tail-mutated receptors remain sequestered. Thus, the direct binding of the ␤2AR to NSF demonstrates how, after internalization, the fate of a receptor is reliant on a specific interaction with a component of the cellular membrane-trafficking machinery.G protein-coupled receptors (GPCRs) 1 are a family of integral plasma membrane proteins that transduce signals into cells from diverse extracellular ligands. An almost universal feature of these receptors is their ability to be desensitized in response to prolonged exposure to ligand (1). This is achieved mainly by the action of GPCR kinases that only phosphorylate agonist-occupied receptor molecules, allowing ␤arrestin molecules to then bind and physically interdict further coupling of the receptor to heterotrimeric G proteins (2).Receptor desensitization is usually accompanied by the rapid internalization of the receptor molecules. This serves two main purposes. Firstly, it allows a cell to resensitize its responsiveness to a ligand by trafficking the receptors through a series of intracellular vesicular compartments in which they are returned to a naïve state (i.e. dephosphorylated) before recycling them back to the cell surface (3-6). Secondly, by trafficking the receptors to alternative vesicular structures in which they are degraded, a cell can permanently reduce the receptor density in the plasma membrane and thus diminish its responsiveness to subsequent exposures to the ligand (7-9).The proportion of a GPCR that is targeted for recycling or degradation varies greatly between receptor types, as do the rates with which these two processes occur. Some GPCRs, including the ␤2 adrenergic receptor (␤2AR), are rapidly recycled back to the cell surface within minutes of being internalized (3, 10 -12). Other receptors are detained within the cell for much longer...

Recently, we identified a GTPase-activating protein for the ADP ribosylation factor family of small GTP-binding proteins that we call GIT1. This protein initially was identified as an interacting partner for the G protein-coupled receptor kinases, and its overexpression was found to affect signaling and internalization of the prototypical ␤2-adrenergic receptor. Here, we report that GIT1 overexpression regulates internalization of numerous, but not all, G protein-coupled receptors. The specificity of the GIT1 effect is not related to the type of G protein to which a receptor couples, but rather to the endocytic route it uses. GIT1 only affects the function of G protein-coupled receptors that are internalized through the clathrin-coated pit pathway in a ␤-arrestin-and dynamin-sensitive manner. Furthermore, the GIT1 effect is not limited to G proteincoupled receptors because overexpression of this protein also affects internalization of the epidermal growth factor receptor. However, constitutive agonist-independent internalization is not regulated by GIT1, because transferrin uptake is not affected by GIT1 overexpression. Thus, GIT1 is a protein involved in regulating the function of signaling receptors internalized through the clathrin pathway and can be used as a diagnostic tool for defining the endocytic pathway of a receptor.

No abstract