Cannabinoid (CB) type-1 receptors (CB1) in the central nervous system (CNS) mediate the psychoactive effects of delta-9-tetrahydrocannabinol, the major active constituent in marijuana. CB1 receptors also mediate many effects of the lipid-derived endogenous cannabinoids (endocannabinoids). This endocannabinoid system plays important roles in regulating motor activity and coordination, short-term memory, pain perception, metabolic homeostasis and drug reward and craving. CB1 receptors can be regulated by post-translational modification and protein-protein interactions, which can alter functional activity, cellular localization and expression levels of these receptors. These processes play a role in limiting the duration of action of CB agonists and in the development of tolerance or dependence upon repeated administration of CB agonists. The proposed project will investigate the function of a newly discovered CB receptor-interacting protein, CRIP1a, which binds to the distal C-terminus of CB1 receptors and attenuates constitutive (basal) activity of these receptors. Preliminary findings also suggest that CRIP1a can alter agonist-induced CB1 signaling in a ligand- and signaling pathway-dependent manner. Preliminary data indicate that CRIP1a can inhibit agonist- induced downregulation or desensitization of CB1 receptors, and that CRIP1a is co-localized with CB1 receptors, particularly in CNS glutamatergic neurons. The following specific aims are proposed to investigate the function of CRIP1a: 1) develop novel cell lines and siRNA constructs as tools to determine the effects of CRIP1a on acute and chronic activation of CB1 receptors and 2) develop a CRIP1a knockout mouse line as a novel tool to investigate effects of CRIP1a on physiological function, behavior and CB pharmacology in vivo. Biochemical and cell imaging approaches will be used to determine effects of co-expression or siRNA-mediated knockdown of CRIP1a in cell models on CB1 receptor-mediated G-protein association (co-immunoprecipitation) and activation (GTP3S binding), and interaction with the regulatory protein 2-arrestin. Effects of CRIP1a on CB1 receptor desensitization, downregulation and internalization will then be examined in these cell models. A CRIP1a gene knockout mouse line will be created using a "flox" approach. Knockout mice will be subjected to basic health assessment and in vivo phenotyping, followed by determination of effects of the knockout on the pharmacological potency of CB agonists in tests of hypothermia, hypolocomotion, catalepsy and antinociception. Anatomical and biochemical studies will then be conducted to determine effects of CRIP1a knockout on CB1 receptor levels, G-protein activation and cellular localization in the CNS. These studies will provide valuable data concerning the role of CRIP1a in the regulation of CB1 receptor-mediated signal transduction associated with functional responses in animals. This work will provide novel target leads for development of drugs that selectively regulate the activity of CB1 receptors for the treatment of drug addiction and other diseases in which the endocannabinoid system is a critical modulatory component. PUBLIC HEALTH RELEVANCE: CB1 cannabinoid receptors mediate many of the effects of marijuana and interact with naturally occurring marijuana-like substances in the brain. This system is important in the regulation of appetite, pain perception, memory, movement and coordination, and seems to play a role in the rewarding effects of several addictive drugs. The proposed project would study a newly discovered protein, called CRIP1a, which interacts with CB1 receptors and appears to modulate their function. These studies will investigate the role of CRIP1a in the regulation of CB1 receptors using genetically modified cultured cell lines and mice in which the CRIP1a gene has been inactivated, to increase our understanding of the effects of marijuana in the brain and perhaps provide a novel target for development of drugs that selectively regulate the activity of CB1 receptors.

R21DA025321

2009-04-10

2013-02-28