Endogenous G protein-coupled receptor kinase 6 triggers homologous β-adrenergic receptor desensitization in primary uterine smooth muscle cells

Abstract

We previously reported that G protein-coupled receptor kinase (GRK) may contribute to β-adrenergic receptor (β-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 β-AR desensitization was assessed by an approximately 60% decrease in cAMP production to a subsequent challenge with the β-agonist, isoproterenol. Desensitization was reduced by 36% with a GRK inhibitor, heparin, and by 31% with a protein kinase A inhibitor, H89. Using antibodies known to specifically inhibit either GRK2/3 or GRK4-6 families, we demonstrated that only the GRK4-6 family mediated β-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 (K215RGRK5 and K215RGRK6). Expression of K215RGRK6 increased by approximately 70% the cAMP response to isoproterenol without effect on forskolin stimulation. Conversely, expression of K215RGRK5 or K220RGRK2 had no effect on β-adrenergic signaling. These results strongly suggest that endogenous GRK6 mediate homologous β-AR desensitization in myometrial cells.