Sequential phosphorylation of rhodopsin at multiple sites

Ohguro, Hiroshi; Palczewski, Krzysztof; Ericsson, Lowell H.; Walsh, Kenneth A.; Johnson, Richard S.

doi:10.1021/bi00072a030

Cited by 199 publications

(140 citation statements)

References 27 publications

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“…For rhodopsin (12), the N-formyl peptide receptor (18) as well as the C5a receptor (19), GRK-mediated phosphorylation seems to proceed sequentially. On the contrary, our findings suggest that GRK2 can phosphorylate Ser 404 , Ser 408 , and Ser 410 of the ␣ 1B AR independently (Fig.…”

Section: Discussionmentioning

confidence: 99%

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

Diviani¹,

Lattion²,

Cotecchia³

1997

Journal of Biological Chemistry

117

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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...

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Section: Discussionmentioning

confidence: 99%

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

Diviani¹,

Lattion²,

Cotecchia³

1997

Journal of Biological Chemistry

117

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“…The identity of rhodopsin phosphorylation sites has been determined by analysis of a Cterminal peptide derived from isolated light-activated rod outer segments [68][69][70]. The initial sites of phosphorylation were identified at Ser$$) or Ser$%$.…”

Section: Grk Phosphorylation Sitesmentioning

confidence: 99%

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

Böhm

Grady

Bunnett

1997

Biochemical Journal

476

322

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The large and functionally diverse group of G-protein-coupled receptors includes receptors for many different signalling molecules, including peptide and non-peptide hormones and neurotransmitters, chemokines, prostanoids and proteinases. Their principal function is to transmit information about the extracellular environment to the interior of the cell by interacting with the heterotrimeric G-proteins, and they thereby participate in many aspects of regulation. Cellular responses to agonists of these receptors are usually rapidly attenuated. Mechanisms of signal attenuation include removal of agonists from the extracellular fluid, receptor desensitization, endocytosis and downregulation. Agonists are removed by dilution, uptake by transporters and enzymic degradation. Receptor desensitization is mediated by receptor phosphorylation by G-protein receptor kinases and second-messenger kinases, interaction of phosphorylated receptors with arrestins and receptor uncoupling from

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“…Sequential phosphorylation of different residues has been demonstrated in the case of rhodopsin, in which light-dependent phosphorylation occurs first at the serine in position 338, and subsequently at serine 343 and then threonine 336 (4,(32)(33)(34). In the case of the human C5a anaphylatoxin receptor, phosphoamino acid analysis of C5a-dependent phosphorylation showed the maximal stoichiometry to be 6 moles of phosphate/ mole of receptor.…”

Section: Fig 2 Effects Of Substitution Of Serines and Threonines Inmentioning

confidence: 99%

Agonist-induced Desensitization of the μ Opioid Receptor Is Determined by Threonine 394 Preceded by Acidic Amino Acids in the COOH-terminal Tail

Pak¹,

O’Dowd²,

George³

1997

Journal of Biological Chemistry

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To identify the structural determinants necessary for opioid receptor desensitization, we serially ablated potential phosphorylation sites in the carboxyl tail of the receptor and examined their effects on [D-Ala 2 ,N-MePhe 4 ,Gly-ol 5 ]enkephalin (DAMGO)-induced desensitization. First, we replaced Thr 394 with alanine (T394A) and stably expressed this mutant receptor in Chinese hamster ovary cells. The T394A receptor did not desensitize after 1 h of treatment with DAMGO, indicating that Thr 394 is required for agonist-induced early desensitization. To test whether Thr 394 was the only residue necessary, we investigated the importance of 7 potential phosphorylation sites between residues 363 and 383, which were all replaced by alanines with the Thr 394 maintained. This mutant (AT) showed partial loss of desensitization (30%), which was attributable to the Ala mutation at Thr 383 , since complete desensitization was achieved by restoring Thr 383 (ATT). These results suggest that Thr 394 is the primary recognition site for G protein-coupled receptor kinases, but Thr 383 is also required for complete agonist-induced desensitization. The specificity of Thr 394 as the primary initiation site appears to be dependent on the preceding acidic amino acid stretch, because in a mutant in which glutamic acid residues at 388, 391, and 393 were replaced by glutamines (EQ), agonist-induced desensitization was completely abolished, identical to the T394A mutant.Desensitization is defined as loss of a biological response to a ligand despite its continuous presence. This phenomenon of desensitization is observed with almost every receptor, and some of the mechanisms involved in desensitization of G protein-coupled receptors have been well characterized using the ␤-adrenergic receptor (␤AR) 1 and rhodopsin. For these receptors, the primary mechanism of desensitization appears to be receptor phosphorylation mediated by G protein-coupled receptor kinases (GRKs). Since these kinases have the unique feature of phosphorylating receptors only when they are in an active or a ligand-bound conformation, GRK-mediated phosphorylation is considered to be the main mechanism for agonist-induced desensitization. These kinases have been shown to phosphorylate a number of G protein-coupled receptors including the ␤AR (1, 2), rhodopsin (3, 4), ␣ 2 -adrenergic receptor (␣ 2 AR) (5, 6), platelet-activating factor receptor (7), thrombin receptor (8), and C5a anaphylatoxin receptor (9).Endogenous opioids play an important role in a variety of physiological processes through pharmacologically distinct, three major receptor subtypes that are coupled to inhibitory G proteins (10). Although the acute action of opioids can induce a number of beneficial effects, chronic use of opioids produces tolerance and dependence (11, 12), which are among the major factors limiting the clinical use of opioids. The molecular mechanisms underlying these phenomena are poorly understood, and receptor desensitization has been implicated as a possible mechanism.It has been sho...

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Sequential phosphorylation of rhodopsin at multiple sites

Cited by 199 publications

References 27 publications

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

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

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

Agonist-induced Desensitization of the μ Opioid Receptor Is Determined by Threonine 394 Preceded by Acidic Amino Acids in the COOH-terminal Tail

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