While well defined pharmacologically, it has been difficult to show that marijuana and its principle psychoactive ingredient, delta9-THC, mediate their multitude of effects via a single specific receptor. The recent cloning of a cannabinoid receptor affords the opportunity to examine the binding properties of these receptors in transfected cell lines and to compare the structure-activity relationships (SAR) of binding with those of whole animal pharmacology. The SAR of binding of this and other putative cannabinoid receptors need to be correlated with that of whole animal pharmacology to verify that the site is relevant to cannabinoids and to determine which pharmacological effects are associated with it. In this project, the sequence information obtained from this cloned cannabinoid receptor will be used to search for homologous receptors in order to determine their relationship to cannabinoid pharmacology. Putative receptor cDNAs will initially be cloned from a rat brain library using a probe based on sequences unique to the published cannabinoid receptor. The tissue distribution (in the rat brain and periphery) of the clones will be assessed by RNA blot analysis and in situ hybridization. This distribution will be compared with that of receptor binding/autoradiography in the whole animal. The putative receptors will be transfected into mammalian cell lines (which don't contain cannabinoid receptors) and their binding characteristics determined using multiple cannabinoid binding assays. The binding data will be compared to the established SAR obtained in whole brain homogenates. In addition, the second messenger properties of these receptors will be evaluated to determine if there are differences between subtypes. Selective mutations will be introduced in regions of the receptor cDNAs believed to be critical to receptor recognition and second messenger function. The mutant cDNAs will be transfected and their ligand binding and coupling characteristics assessed. Finally, homologous receptors will be cloned from a human library and assessed by transfection into mammalian cell lines. Comparisons between human and rat receptor properties could establish the functional significance of the receptors with respect to cannabinoid psychoactivity in humans as well as pharmacological responses in rats. Insight into cannabinoid receptors may lead to a better understanding of cannabinoid abuse in humans.
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