Receptor class desensitization of leukocyte chemoattractant receptors.

The human N-formylpeptide receptor (FPR) represents one of the most thoroughly studied leukocyte chemoattractant receptors. Despite this, little is known about the molecular mechanisms involved in the activation and desensitization of this receptor. To assess the role of phosphorylation in receptor function, U937 promonocytic cells were stably transfected to express the recombinant human FPR. Three mutant forms of the FPR lacking specific serine and threonine residues in the receptor C terminus were studied with respect to activation and desensitization. Replacement of all 11 serine and threonine residues within the C terminus by alanine and glycine residues (⌬ST) resulted in a receptor capable of ligand binding and G protein activation similar to the wild-type receptor. However, whereas the wild-type FPR was phosphorylated on both serine and threonine residues upon exposure to agonist and displayed a significantly reduced ability to stimulate G protein-mediated GTP hydrolysis upon subsequent exposure to agonist, ⌬ST demonstrated a complete lack of phosphorylation and displayed little alteration in its ability to stimulate G protein-mediated GTP hydrolysis upon a subsequent exposure to agonist. In addition to desensitization of G protein-mediated GTP hydrolysis, calcium mobilization was assayed to test whether desensitization occurred at a site distal to G protein activation. However, as observed with G protein activation, ⌬ST underwent no desensitization of the calcium mobilization response upon a second exposure to agonist. To define more precisely the role of specific serine and threonine residues, two additional mutants were analyzed. Neutrophils possess a large number of cell-surface G proteincoupled receptors that respond to structurally diverse ligands such as N-formylpeptides, complement components C5a and C3a, platelet-activating factor, and chemokines such as IL-8 1 (1). Receptor activation results in the stimulation of phospholipases, the mobilization of intracellular calcium, and the activation of a multitude of protein kinases culminating in functions such as chemotaxis, phagocytosis, superoxide production, and degranulation. Following an initial exposure to ligand, resulting in transient cell activation, neutrophils rapidly become unresponsive to continued or subsequent stimulation. This process of cellular acquiescence in the presence of agonist is termed desensitization and has been characterized for many hormonal (2) and neurotransmitter (3) receptors. Although the complex mechanisms involved in this process are poorly characterized, one of the early events has been suggested to involve receptor phosphorylation (4).G protein-coupled receptor kinases are a family of protein kinases that rapidly phosphorylate seven transmembrane receptors in a ligand-dependent manner (5-7). Following phosphorylation and the possible association with accessory proteins, such as arrestin, receptors are no longer capable of effectively activating G proteins, a process termed homologous desensitization (8). G protein-...

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Desensitization of N-Formylpeptide Receptor-mediated Activation Is Dependent upon Receptor Phosphorylation

Prossnitz

1997

“…The mutated DNA fragments obtained were digested with BssHII and ApaI and cloned into the expression vector pRK5 (24) containing the ␣ 1B AR cDNA. Recombinant clones were isolated and sequenced by cycle sequencing using Exo Ϫ Pfu polymerase (Stratagene).…”

Section: Methodsmentioning

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

117

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...

“…nomenon termed class desensitization (38,39). Recently, it was shown that PLC␤3 was phosphorylated both by PKC and PKA mediated pathways (25) and PLC␤2 was phosphorylated by PKA (24).…”

Section: Fig 4 Desensitization Of Cxcr4mentioning

confidence: 99%

Regulation of Human Chemokine Receptors CXCR4

Haribabu

Richardson

Fisher

et al. 1997

261

100

Members of the chemokine receptor family CCR5 and CXCR4 have recently been shown to be involved in the entry of human immunodeficiency virus (HIV) into target cells. Here, we investigated the regulation of CXCR4 in rat basophilic leukemia cells (RBL-2H3) stably transfected with wild type (Wt CXCR4) or a cytoplasmic tail deletion mutant (⌬Cyto CXCR4) of CXCR4. The ligand, stromal cell derived factor-1 (SDF-1) stimulated higher G-protein activation, inositol phosphate generation, and a more sustained calcium elevation in cells expressing ⌬Cyto CXCR4 relative to Wt CXCR4. SDF-1 and phorbol 12-myristate 13-acetate (PMA), but not a membrane permeable cAMP analog induced rapid phosphorylation as well as desensitization of Wt CXCR4. Phosphorylation of ⌬Cyto CXCR4 was not detected under any of these conditions. Despite lack of receptor phosphorylation, calcium mobilization by SDF-1 in ⌬Cyto CXCR4 cells was partially desensitized by prior treatment with SDF-1. Of interest, the rapid release of calcium was inhibited without affecting the sustained calcium elevation, indicating independent regulatory pathways for these processes. PMA completely inhibited phosphoinositide hydrolysis and calcium mobilization in Wt CXCR4 but only partially inhibited these responses in ⌬Cyto CXCR4. cAMP also partially inhibited these responses in both Wt CXCR4 and ⌬Cyto CXCR4. SDF-1, PMA, and cAMP caused phosphorylation of phospholipase C␤3 in Wt and ⌬Cyto CXCR4 cells. Both SDF-1 as well as PMA induced rapid internalization of Wt CXCR4. SDF-1 but not PMA induced internalization of ⌬Cyto CXCR4 albeit at reduced levels relative to Wt CXCR4. These results indicate that signaling and internalization of CXCR4 are regulated by receptor phosphorylation dependent and independent mechanisms. Desensitization of CXCR4 signaling, independent of receptor phosphorylation, appears to be a consequence of the phosphorylation of phospholipase C␤3.Chemokines are a group of proteins that mediate directed migration and activation of leukocytes (1). These have been classified into two main families, CXC or CC-chemokines. Two newly identified proteins define additional groups of C and CX3C chemokines based on the position of conserved cysteines (2, 3). Both CC and CXC chemokines bind to seven transmembrane G-protein-coupled receptors which transduce signals through heterotrimeric G-proteins (4). Several recent studies showed that chemokines play a significant role in human immunodeficiency virus (HIV-1) 1 infection and that the chemokine receptors CCR5 and CXCR4 along with CD4 act as major co-receptors for the macrophage tropic and T-cell tropic HIV-1 strains entry, respectively, into target cells (5-10). While the C-C chemokines such as MIP1␣, MIP1␤, and regulated upon activation, normal T expressed and secreted can activate CCR5 the CXC chemokine SDF-1 is the ligand for CXCR4 (11-13). Recently, CD4 independent infection by HIV-2 was shown to be mediated by CXCR4 (14). HIV-1 envelope glycoproteins interact with chemokine receptors in a CD4-dependent and in one case...