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.