Characterization of the Phosphorylation Sites Involved in G Protein-coupled Receptor Kinase- and Protein Kinase C-mediated Desensitization of the α1B-Adrenergic Receptor

Abstract

Catecholamines as well as phorbol esters can induce the phosphorylation and desensitization of the ␣ 1B -adrenergic receptor (␣ 1B AR). In this study, phosphoamino acid analysis of the phosphorylated ␣ 1B AR revealed that both epinephrine-and phorbol ester-induced phosphorylation predominantly occurs at serine residues of the receptor. The findings obtained with receptor mutants in which portions of the C-tail were truncated or deleted indicated that a region of 21 amino acids (393-413) of the carboxyl terminus including seven serines contains the main phosphorylation sites involved in agonist-as well as phorbol ester-induced phosphorylation and desensitization of the ␣ 1B AR. To identify the serines invoved in agonist-versus phorbol ester-dependent regulation of the receptor, two different strategies were adopted, the seven serines were either substituted with alanine or reintroduced into a mutant lacking all of them. Desensitization is a general regulatory phenomenon of G protein-coupled receptors resulting in the attenuation of the receptor-mediated response. Two major patterns of desensitization referred to as homologous and heterologous desensitization can be distinguished. Homologous desensitization is defined as a rapid loss of responsiveness for a receptor repeatedly exposed to its specific agonist, whereas in heterologous desensitization stimulation of a receptor by an agonist can attenuate the response mediated by other receptors eliciting similar cellular effects (1).In the G protein-coupled receptor family (2), receptor desensitization has been extensively characterized for rhodopsin mediating phototransduction in retinal rod cells and for the ␤ 2 -adrenergic receptor (␤ 2 AR) 1 which mediates catecholamineinduced stimulation of adenylyl cyclase. The second messengerdependent cAMP-dependent protein kinase can phosphorylate and desensitize the ␤ 2 AR both in response to its agonist as well as to other agents increasing the cellular content of cAMP. On the other hand, a prominent role in homologous desensitization of rhodopsin and ␤ 2 AR is played by the second messengerindependent rhodopsin kinase (3) and ␤-adrenergic receptor kinase (␤ARK) (4), respectively. Once the receptor is occupied by the agonist, it is recognized by the kinase and becomes phosphorylated. The subsequent uncoupling of the receptor and G protein is then mediated by arrestin proteins, which specifically bind to the phosphorylated receptor (5, 6). Rhodopsin kinase and ␤ARK are members of the newly discovered family of G protein-coupled receptor kinases (GRK) (7). These protein kinases have the unique ability to recognize and phosphorylate their G protein-coupled receptor substrates predominantly in their active (i.e. agonist-occupied) conformations. Recently, we have provided evidence that the ␣ 1B AR coupled to Gq-mediated activation of phospholipase C can be phosphorylated by at least two types of protein kinases, a protein kinase C (PKC) upon its stimulation by phorbol esters (8) and protein kinases belonging to the GRK family o...

Threonine 308 within a Putative Casein Kinase 2 Site of the Cytoplasmic Tail of Leukotriene B4 Receptor (BLT1) Is Crucial for Ligand-induced, G-protein-coupled Receptor-specific Kinase 6-mediated Desensitization

“…In another system, epinephrine-induced phosphorylation of the ␣ 1B -adrenergic receptor by GRK6 did not correlate with attenuation of receptor signaling in COS-7 cells (63). However, in several cases, clusters of serine and threonine residues have been mapped for their contribution to desensitization (64,65). As with the ␤ 2 -adrenergic receptor (65), BLT1 desensitization may not be dependent on a single residue.…”

The Adaptor Complex 2 Directly Interacts with the α1b-Adrenergic Receptor and Plays a Role in Receptor Endocytosis

“…Interacting with the ␣ 1b -AR-It was previously reported that the C-tail of the ␣ 1b -AR plays an important role in the regulation of receptor desensitization and internalization (19,22,23). However, the molecular players involved in these processes are not fully characterized.…”

C-Terminal Tail Phosphorylation of N-Formyl Peptide Receptor: Differential Recognition of Two Neutrophil Chemoattractant Receptors by Monoclonal Antibodies NFPR1 and NFPR2

“…The human receptor is in low abundance and has yet to be purified (4) in quantities suitable for biochemical or structural characterization. As most GPCRs thus far examined, FPR activity is exquisitely regulated by cellular kinases (5)(6)(7)(8), surface reorganization (9,10), internalization and recycling (11) and interaction with adaptor proteins such as arrestins (12) and possibly others (13). These forms of regulation may rely on receptor posttranslational modifications, conformational changes, and/or spatial accessibility to signal transduction partners.…”