Defense Date

2009

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Pharmacology & Toxicology

First Advisor

Dana Selley

Abstract

EFFECTS OF CANNABINOID RECEPTOR INTERACTING PROTEIN (CRIP1a) ON CANNABINOID (CB1) RECEPTOR FUNCTION. By Tricia Hardt Smith, B.S., M.S. A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University Virginia Commonwealth University, 2009. Major Director: Dana E. Selley, Ph.D., Department of Pharmacology and Toxicology This dissertation examines modulation of cannabinoid CB1 receptor function by Cannabinoid Receptor Interacting Protein (CRIP1a), a novel protein that binds the C-terminus of CB1 receptors. In Human embryonic kidney cells expressing human CB1 receptors (hCB1-HEK) and hCB1-HEK cells stably co-expressing CRIP1a (hCB1-HEK-CRIP1a), quantitative immunoblotting revealed a CRIP1a/CB1 molar ratio of 5.4 and 0.37, respectively, with no difference in CB1 receptor expression. To test the hypothesis that CRIP1a modulates CB1 receptor signaling, G-protein and effector activity were examined with and without full, partial and inverse agonists. [35S]GTPgS binding, which measures G-protein-coupled receptor (GPCR)-mediated G-protein activation, showed that CRIP1a inhibited constitutive CB1 receptor activity, as indicated by the decreased effect of the inverse agonist SR141716A. CRIP1a also decreased CB1 receptor-mediated G-protein activation by high efficacy agonists, whereas moderate and low efficacy agonists were unaffected. In experiments varying Na+ concentration, CRIP1a decreased spontaneous G-protein activation at low Na+ concentrations, where spontaneous GPCR activity is highest. This effect was eliminated by pertussis toxin pre-treatment, indicating that CRIP1a only inhibits GPCR-mediated activity. To determine whether CRIP1a modulates receptor adaptation, hCB1-HEK (±CRIP1a) cells were pretreated with WIN or THC. Both ligands desensitized CB1 receptor-mediated G-protein activation, but desensitization was unaffected by CRIP1a. In contrast, CRIP1a attenuated downregulation of CB1 receptor binding sites by WIN, but not THC. Downstream, CRIP1a attenuated constitutive CB1 receptor-mediated inhibition of cAMP, as indicated by elimination of SR141716A-stimulated cAMP, without affecting agonist-induced cAMP inhibition. Constitutive inhibition was not due to endocannabinoids because LC-ESI-MS-MS did not detect endocannabinoids in hCB1-HEK (±CRIP1a) cells. To determine whether effects of CRIP1a were conserved among cell types, Chinese Hamster Ovary cells expressing CB1 receptors were stably co-transfected with CRIP1a, and had a CRIP1a/CB1 receptor molar ratio of 15 and 1900 with and without CRIP1a over-expression, respectively. In this model, CRIP1a inhibited constitutive CB1 receptor-mediated G-protein activity, but activation by agonists was enhanced, suggesting CRIP1a effects were dependent on stoichiometry of CRIP1a/CB1 receptor or cell type. Overall, these results indicate that CRIP1a decreases constitutive CB1 receptor activity, modulates agonist efficacy, and inhibits CB1 receptor downregulation, in a ligand- and cellular environment-dependent manner.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

December 2009

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