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.

Protein structure predication is a field of computational molecular modeling with an enormous potential for improvement. Side-chain geometry prediction is a critical component of this process that is crucial for computational protein structure predication as well as crystallographers in refining experimentally determined protein crystal structures. The cornerstone of side-chain geometry prediction are side-chain rotamer libraries, usually obtained through exhaustive statistical analysis of existing protein structures. Little is known, however, about the driving forces leading to the preference or suitability of one rotamer over another. Construction of 3D hydropathic interaction maps for nearly 30,000 tyrosines extracted from the PDB reveals their environments, in terms of hydrophobic and polar (collectively “hydropathic”) interactions. Using a unique 3D similarity metric, these environments were clustered with k-means. In the ϕ, ψ region (–200° < ϕ < –155°; –205° < ψ < –160°) representing 631 tyrosines, clustering reduced the set to 14 unique hydropathic environments, with most diversity arising from favorable hydrophobic interactions. Polar interactions for tyrosine include ubiquitous hydrogen bonding with the phenolic OH and a handful of unique environments surrounding the backbone. The memberships of all but one of the 14 environments are dominated by a single χ1/χ2 rotamer. Each tyrosine residue attempts to fulfill its hydropathic valence. Structural water molecules are thus used in a variety of roles throughout protein structure. A second project involves elucidating the 3D structure of CRIP1a, a cannabinoid 1 receptor (CB1R) binding protein that could provide information for designing small molecules targeting the CRIP1a-CB1R interaction. The CRIP1a protein was produced in high purity. Crystallization experiments failed, both with and without the last 9 or 12 amino acid peptide of the CB1R C-terminus. Attempts were made to use NMR for structure determination; however, the protein precipitated out during data acquisition. A model was thus built computationally to which the CB1R C-terminus peptide was docked. HINT was used in selecting optimum models and analyzing interactions involved in the CRIP1a-CB1R complex. The final model demonstrated key putative interactions between CRIP1a and CB1R while also predicting highly flexible areas of the CRIP1a possibly contributing to the difficulties faced during crystallization.