G-protein-coupled receptor kinase 3- and protein kinase C-mediated desensitization of the PACAP receptor type 1 in human Y-79 retinoblastoma cells

Abstract

Pituitary adenylyl cyclase-activating polypeptide (PACAP) receptor type 1 (PAC 1) signaling and desensitization were investigated in human retinoblastoma Y-79 cells. Concentration-dependent stimulation of cAMP accumulation was observed in Y-79 cells incubated for 30 min with PACAP38, PACAP27, or VIP (10 −12 to 10 −6 M). The following EC 50 values were calculated: PACAP38, 24±3 pM; PACAP27, 99±8 pM; and VIP, 29±3 nM. Homologous desensitization of PAC 1 receptors in Y-79 cells pretreated with 10 nM PACAP38 or PACAP27 for 60 min was characterized by a 30–50% reduction in PACAP-stimulated cAMP accumulation ( p<0.0001) and a two- to fivefold rightward shift in EC 50 values ( p<0.0001). PAC 1 receptor desensitization was not accompanied by a reduction in PAC 1 mRNA expression. We concluded that the desensitizing effect of PACAP38 was homologous because neither corticotropin-releasing factor- nor (−)-isoproterenol-stimulated cAMP accumulation was altered by PACAP38 preincubation. Pretreating Y-79 cells with the protein kinase A (PKA) inhibitor H89 failed to inhibit homologous PAC 1 receptor desensitization. Similarly, pretreating Y-79 cells with the protein kinase C (PKC) inhibitors staurosporine or bisindolylmaleimide failed to alter homologous PAC 1 receptor desensitization. Although activation of PKA by dibutyryl cAMP or forskolin did not desensitize PAC 1 receptors, direct activation of PKC by PMA heterologously desensitized PAC 1 receptors, reducing cAMP accumulation 34.2±2.2% ( p<0.001). Using RT–PCR, mRNA levels for G-protein-coupled receptor kinase 3 (GRK3), but not GRK2, were found to increase 2.2- to 4.8-fold in Y-79 cells exposed to PACAP38 for 10 min to 24 h ( p<0.001). PAC 1 receptor desensitization decreased 72.5±4.3% ( p<0.001) in Y-79 cells transfected with a GRK3 antisense cDNA construct that also reduced GRK3 protein expression 48.5±7.9% ( p<0.0005). These experiments demonstrate that GRK3 plays an important role in the homologous desensitization of retinoblastoma PAC 1 receptors, whereas PKC, but not PKA, contributes to the heterologous desensitization of retinoblastoma PAC 1 receptors.