G-protein-coupled receptor kinase 3- and protein kinase C-mediated desensitization of the PACAP receptor type 1 in human Y-79 retinoblastoma cells.
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.
Dautzenberg
F M
FM
Pharma Division, Preclinical Research, F. Hoffmann-La Roche Ltd, CH-4070, Basel, Switzerland.
Hauger
R L
RL
D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D017398:Alternative Splicing; D003346:Corticotropin-Releasing Hormone / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D016373:DNA, Antisense / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D054770:G-Protein-Coupled Receptor Kinase 3; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D009479:Neuropeptides / Q000494:pharmacology; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D011943:Receptors, Adrenergic, beta / Q000378:metabolism; D018019:Receptors, Corticotropin-Releasing Hormone / Q000378:metabolism; D051236:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I; D011979:Receptors, Pituitary Hormone / Q000235:genetics / Q000378:metabolism*; D012175:Retinoblastoma / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D014407:Tumor Cells, Cultured; D014660:Vasoactive Intestinal Peptide / Q000494:pharmacology; D000099052:CRF Receptor, Type 1
C495401:ADCYAP1 protein, human; D000318:Adrenergic beta-Agonists; D016373:DNA, Antisense; D004791:Enzyme Inhibitors; D009479:Neuropeptides; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D012333:RNA, Messenger; D011943:Receptors, Adrenergic, beta; D018019:Receptors, Corticotropin-Releasing Hormone; D051236:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I; D011979:Receptors, Pituitary Hormone; D014660:Vasoactive Intestinal Peptide; D000099052:CRF Receptor, Type 1; D003346:Corticotropin-Releasing Hormone; D000242:Cyclic AMP; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D054770:G-Protein-Coupled Receptor Kinase 3; C517848:GRK3 protein, human