Abstract 2371 Cannabinoid receptor interacting protein 1a (CRIP1a) potential as a cargo carrier for lipidated G alpha subunits

Introduction: The cannabinoid receptor interacting protein 1a (CRIP1a) is a neuronal protein implicated in the human endocannabinoid system (ECS). CRIP1a interacts with the C-terminus of the neuromodulatory CB1 cannabinoid receptor (CB1R) abundant at axon termini. CB1R is a G protein coupled receptor (GPCR) which predominantly transmits signals via heterotrimeric G proteins of the i/o subtype. The CB1R-mediated G protein activation can be attenuated by CRIP1a overexpression via an unknown mechanism. To elucidate this mechanism, we solved the CRIP1a crystal structure and discovered CRIP1a exhibits structural homology to a family of lipidated-cargo carriers. Notably, heterotrimeric G protein subunits alpha (Gα) and gamma (Gγ) are invariably lipidated. The type of lipid moiety, number, location, and reversibility depend upon subunit subtype. In addition to serving as a plasma membrane anchor, these lipid modifications influence G protein localization and signaling. Objective: We are investigating the potential for CRIP1a to function as a lipidated-cargo carrier for heterotrimeric G protein subunits as a mechanism for CRIP1a-mediated CB1R signaling regulation. Methods: Recombinant protein constructs were individually purified from E. coli. Peptides mimicking lipidated candidate proteins were synthesized and linked to fluorescein isothiocyanate (FITC). Binding between recombinant proteins was qualitatively assessed using affinity pull-downs. Binding affinities between the FITC-peptide mimics and recombinant protein constructs were determined using fluorescence polarization. Results: Recombinant CRIP1a was capable of binding N-terminally lipidated Gα peptide-mimics, but not the C-terminally lipidated Gγ mimics. Binding affinity was sensitive to the length, primary sequence, and lipidation state of the peptide. CRIP1a was capable of binding to full length Gαi1, a Gα subtype activated by CB1R. Conclusions and Implications: Our data support a role for CRIP1a as a lipidated-cargo carrier targeting Gα subunits, particularly Gαi subtypes which are activated by CB1R. With such a role, CRIP1a could influence neuronal CB1R signaling by altering the available pool of Gα subunits accessible to form the heterotrimer and be activated for intracellular signaling. The relationship between CRIP1a-Gα binding and CRIP1a-CB1R C-terminus complexation requires further research. Funding by the Louis Argenta Physician-Scientist Fellowship and the National Institute of Health (NIH) (Grant no. R01-DA042157).