Endogenous G protein-coupled receptor kinase 6 triggers homologous beta-adrenergic receptor desensitization in primary uterine smooth muscle cells.
We previously reported that G protein-coupled receptor kinase (GRK) may contribute to beta-adrenergic receptor (beta-AR) uncoupling occurring just before parturition in rat uterine muscle (myometrium). To identify the GRK involved, we set up in this study a primary cell culture retaining the morphological and functional characteristics of myometrial tissue as well as the in vivo pattern of GRK expression (GRK2, GRK5, and GRK6). In this model, homologous beta-AR desensitization was assessed by an approximately 60% decrease in cAMP production to a subsequent challenge with the beta-agonist, isoproterenol. Desensitization was reduced by 36% with a GRK inhibitor, heparin, and by 31% with a protein kinase A in-hibitor, H89. Using antibodies known to specifically inhibit either GRK2/3 or GRK4-6 families, we demonstrated that only the GRK4-6 family mediated beta-AR desensitization. To discriminate between endogenous GRK5 and GRK6, we attempted to inhibit their action by introducing, into myometrial cells, kinase-dead dominant-negative mutants ((K215R)GRK5 and (K215R)GRK6). Expression of (K215R)GRK6 increased by approximately 70% the cAMP response to isoproterenol without effect on forskolin stimulation. Conversely, expression of (K215R)GRK5 or (K220R)GRK2 had no effect on beta-adrenergic signaling. These results strongly suggest that endogenous GRK6 mediate homologous beta-AR desensitization in myometrial cells.
Simon
Violaine
V
Laboratoire de Physiologie et Physiopathologie, Unité Mixte de Recherche 7079, Université P & M Curie, 4 place Jussieu, 75252 Paris Cedex 05, France.
Robin
Marie-Thérèse
MT
Legrand
Chantal
C
Cohen-Tannoudji
Joëlle
J
D000818:Animals; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D005260:Female; D054774:G-Protein-Coupled Receptor Kinase 5; D054768:G-Protein-Coupled Receptor Kinases; D015971:Gene Expression Regulation, Enzymologic; D009130:Muscle, Smooth / Q000166:cytology / Q000201:enzymology*; D016296:Mutagenesis; D009215:Myometrium / Q000166:cytology / Q000201:enzymology*; D010641:Phenotype; D011247:Pregnancy; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D051552:beta-Adrenergic Receptor Kinases
D011943:Receptors, Adrenergic, beta; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D054774:G-Protein-Coupled Receptor Kinase 5; D054768:G-Protein-Coupled Receptor Kinases; C089176:G-protein-coupled receptor kinase 6; C517897:Grk5 protein, rat