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J Biol Chem, Vol. 273, Issue 12, 6756-6762, March 20, 1998
From the Howard Hughes Medical Institute, Departments of Cell
Biology and Medicine, Divisions of Gastroenterology and Cardiology,
Duke University Medical Center, Durham, North Carolina 27710
The secretin receptor is a member of a
structurally distinct class of G protein-coupled receptors designated
as Class II. The molecular mechanisms of secretin receptor signal
termination are unknown. Using transiently transfected HEK 293 cells
expressing the secretin receptor, we investigated its mechanisms of
desensitization. Binding of [125I]-secretin to
plasma membranes of receptor-expressing cells was specific, with a
Kd of 2 nM. Secretin evoked an increase in cellular cAMP with an EC50 of 0.4 nM. The
response was maximal by 20 min and desensitized rapidly and completely.
Immunoprecipitation of a functional, N-terminal epitope-tagged secretin
receptor was used to demonstrate agonist-dependent receptor
phosphorylation, with an EC50 of 14 nM.
Pretreatment with protein kinase A or C inhibitors failed to alter
secretin-stimulated cAMP accumulation. G protein-coupled receptor
kinases (GRKs) are known to be involved in the desensitization of Class
I G protein-coupled receptors; therefore, the effect of cotransfection
of GRKs on secretin-stimulated cAMP signaling and phosphorylation was
evaluated. GRKs 2 and 5 were the most potent at augmenting
desensitization, causing a 40% reduction in the maximal cAMP response
to secretin. GRK 5 also caused a shift in the EC50 to the
right (p < 0.05). GRK 4 and GRK 6 did not alter
dose-dependent signaling, and GRK 3 was intermediate in
effect. Receptor phosphorylation correlated with desensitization for
each GRK studied, whereas second messenger-dependent kinase
phosphorylation appeared to be less important in secretin receptor
signal termination.
We demonstrate agonist-dependent secretin receptor
phosphorylation coincident with profound receptor desensitization of
the signaling function in HEK 293 cells, suggesting a role for receptor phosphorylation in this paradigm. Although GRK activity appears important in secretin receptor desensitization in HEK 293 cells, protein kinases A and C appear to play only a minor role. These results
demonstrate that the GRK-arrestin system regulates Class II G
protein-coupled receptors.
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