Desensitization of N-formylpeptide receptor-mediated activation is dependent upon receptor phosphorylation.
Academic Article
Overview
Research
Identity
Additional Document Info
View All
Overview
abstract
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 (DeltaST) 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, DeltaST 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, DeltaST 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. Replacement either of Ser328, Thr329, Thr331, and Ser332 (mutant A) or of Thr334, Thr336, Ser338, and Thr339 (mutant B) resulted in functional receptors that exhibited approximately 50% the level of phosphorylation following stimulation. Whereas mutant A, like DeltaST, could not be significantly desensitized by exposure to agonist, mutant B exhibited partial desensitization. These results indicate that phosphorylation of the FPR is a necessary and sufficient step in cellular desensitization, that multiple phosphorylation sites are involved, and that redundant desensitization does not occur downstream of G protein activation in the signaling cascade.
