1. To determine the role of G protein-coupled receptor kinases (GRKs) in the regulation of endogenous secretin receptor responsiveness, we have transiently overexpressed both wild-type (WT) and dominant negative mutant (DNM) GRKs in NG108-15 mouse neuroblastoma x rat glioma hybrid cells and investigated the effects of this on agonist-stimulated adenylyl cyclase activity. 2. Overexpression of WT GRK6 selectively inhibited secretin-stimulated cyclic AMP accumulation (fold stimulation of cyclic AMP above basal following 15 min incubation with 100 nM secretin was 12.1+/-2.0 and 6.2+/- 0.8 in control and WT GRK overexpressing cells, respectively) without affecting cyclic AMP responses mediated by the adenosine A(2) receptor agonist 5'-(N-ethylcarboxamido) adenosine (NECA) or the prostanoid-IP receptor agonist iloprost, or the direct activator of adenylyl cyclase, forskolin. On the other hand DNM GRK6 (Lys(215)Arg) overexpression produced the opposite effect--a selective increase in the secretin-stimulated cyclic AMP response was observed in cells overexpressing DNM GRK6 compared to plasmid-transfected cells (fold stimulation of cyclic AMP above basal following 15 min incubation with 100 nM secretin was 12.6+/-2.7 and 29.6+/-5.6 for control and DNM GRK6-overexpressing cells, respectively). 3. Overexpression of WT GRK5 likewise inhibited the secretin-stimulated cyclic AMP response, however, this effect was not as selective as with GRK6, since adenosine A(2) receptor responsiveness was also suppressed by GRK5 overexpression. Unlike DNM GRK6, overexpression of DNM GRK5 failed to modulate secretin or A(2) adenosine receptor signalling suggesting that endogenous GRK5 is unlikely to regulate desensitization of these receptors in NG108-15 cells. 4. Overexpression of WT GRK2 did not affect secretin-stimulated cyclic AMP accumulation. Instead, GRK2 overexpression selectively inhibited A(2) adenosine receptor responsiveness, confirming our previous findings. 5. Together these results suggest a selective role of endogenous GRK6 in regulating secretin receptor responsiveness in NG108-15 cells. In addition, these data indicate that GRKs exert a surprising degree of selectivity in the regulation of natively expressed GPCR responses.