Desensitization of the mouse thromboxane A2 receptor (TP) by G protein-coupled receptor kinases (Grks).
GRKs play a key role in regulating G protein-coupled receptor (GPCR) responsiveness. To investigate the role of GRKs in desensitization of TP, we replaced threonines with favorable phosphorylation motifs for GRKs (positions 226 and 230) with alanine. Mutant and wild-type receptors were expressed in cell culture models and clones expressing similar numbers of receptors were studied. We found that: (1) affinity and specificity of thromboxane A2 (TxA2) binding to mutant TP were identical to the wild-type, (2) replacement of threonines 226 and 230 with alanines delayed the onset of agonist-induced desensitization, and (3) inhibition of endogenous GRK activity with a dominant-negative construct inhibited agonist-induced phosphorylation and enhanced responsiveness of wild-type TP but had little effect on responsiveness of the receptor mutant. These data are consistent with the notion that GRKs contribute to desensitization of TP.
Flannery
Patrick J
PJ
Department of Medicine, Duke University, Durham VA Medical Centers, NC 27710, USA.
Spurney
Robert F
RF
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid / Q000494:pharmacology; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D005920:Glomerular Mesangium; D051379:Mice; D008822:Mice, Transgenic; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D017482:Receptors, Thromboxane / Q000819:agonists / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics; D013912:Threonine / Q000502:physiology; D013928:Thromboxane A2 / Q000378:metabolism; D051552:beta-Adrenergic Receptor Kinases
D017482:Receptors, Thromboxane; D013912:Threonine; D015240:Phorbol 12,13-Dibutyrate; D013928:Thromboxane A2; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D051552:beta-Adrenergic Receptor Kinases