Abstract
Proteins that bind to G protein-coupled receptors have recently been identified as regulators of receptor anchoring and signaling. In this study, actin-binding protein 280 (ABP-280), a widely expressed cytoskeleton-associated protein that plays an important role in regulating cell morphology and motility, was found to associate with the third cytoplasmic loop of dopamine D2 receptors. The specificity of this interaction was originally identified in a yeast two-hybrid screen and confirmed by protein binding. The functional significance of the D2 receptor-ABP-280 association was evaluated in human melanoma cells lacking ABP-280. D2receptor agonists were less potent in inhibiting forskolin-stimulated cAMP production in these cells. Maximal inhibitory responses of D2 receptor activation were also reduced. Further yeast two-hybrid experiments showed that ABP-280 association is critically dependent on the carboxyl domain of the D2 receptor third cytoplasmic loop, where there is a potential serine phosphorylation site (S358). Serine 358 was replaced with aspartic acid to mimic the effects of receptor phosphorylation. This mutant (D2S358D) displayed compromised binding to ABP-280 and coupling to adenylate cyclase. PKC activation also generated D2 receptor signaling attenuation, but only in ABP-containing cells, suggesting a PKC regulatory role in D2-ABP association. A mechanism for these results may be derived from a role of ABP-280 in the clustering of D2 receptors, as determined by immunocytochemical analysis in ABP-deficient and replete cells. Our results suggest a new molecular mechanism of modulating D2 receptor signaling by cytoskeletal protein interaction.
Footnotes
- Received June 28, 1999.
- Accepted November 18, 1999.
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Send reprint requests to: Qun-Yong Zhou, Ph.D., Assistant Professor, Department of Pharmacology, University of California, Irvine, California. E-mail:qzhou{at}uci.edu.
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This work was supported in part by National Institutes of Health Grant MH57889 and a grant from the Alfred E. Sloan Foundation (Q.Y.Z.).
- The American Society for Pharmacology and Experimental Therapeutics
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