The CB1 cannabinoid receptor has been shown to play important physiological roles in the central nervous system, as well as peripherally, and is a target for development of therapeutic medications. To gain insight on the ligand binding site(s) and structural features of activation, we designed and synthesized (Ϫ)-7Ј-isothiocyanato-11-hydroxy-1Ј,1Ј-dimethylheptylhexahydrocannabinol (AM841), a classical cannabinoid affinity label that incorporates an isothiocyanate substituent as an electrophilic reactive group capable of interacting irreversibly with a suitably located and properly oriented nucleophilic amino acid residue at or near the binding site. To obtain evidence for the site of covalent attachment of AM841, C6.47, identified in part by interactive ligand docking, was mutated to serine, alanine, and leucine to reduce or eliminate the nucleophilic character. Wild-type (WT) and mutant CB1 receptors were evaluated for their abilities to recognize a series of cannabinergic ligands. Each bound comparably to WT, excluding C6.47L, which displayed a reduced affinity for It is noteworthy that AM841 was shown to bind irreversibly to WT CB1 but exhibited no covalent attachment with the mutants and behaved as an agonist suggesting irreversible attachment to C6.47 maintains CB1 in its active state. The evidence presented identifies C6.47 as the site of covalent bond formation with AM841 and combined with the binding data fully supports the molecular modeling. These studies present the first report of tandem applications of affinity labeling, site-directed mutagenesis, and interactive ligand docking for CB1.The CB1 and CB2 cannabinoid receptors are relatively new members in the G-protein-coupled receptor (GPCR) superfamily. They have been shown to play important physiological roles and represent targets for development of therapeutic medications. From a pharmacological standpoint, agonist activation of both receptors results in the release of G␣ iproteins, causing a concomitant reduction in intracellular This work has been supported by National Institute on Drug Abuse grants DA05955 (to R.P.P.) DA00355 (to A.D.K.), DA09158, DA03801, DA07215, DA07312 (to A.M.), DA03934, DA00489 (to P.H.R.), DA05274, and DA09978 (to M.E.A.).

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The structural characterization of G-protein coupled receptors (GPCRs) is quite important as these proteins represent a vast number of therapeutic targets involved in drug discovery. However, solving the three-dimensional structure of GPCR has been a significant obstacle in structural biology. A variety of reasons, including their large molecular weight, intricate interhelical packing, as well as their membrane-associated topology, has hindered efforts aimed at their purification. In the absence of pure protein, available in the native conformation, classical methods of structural analysis such as X-ray crystallography and nuclear magnetic resonance spectroscopy cannot be utilized successfully. Alternative methods must therefore be explored to facilitate the structural features involved in drug-receptor interactions. The methods described herein detail the use of covalent probes, or affinity labels, capable of binding covalently to a target GPCR at its binding site(s). Our approach involves the incorporation of a number of reactive moieties in different regions of the ligand molecule each of which is expected to react with different amino acid residues. Information obtained from such work coupled with computer modeling and validated by the use of site-directed mutagenesis of GPCRs allows for three-dimensional mapping of the receptor binding site. It also sheds light on the different possible binding motifs for the various classes of agonists and antagonists and identifies amino acid residues involved with GPCR activation or inactivation.

The progesterone receptor (PR) in the oviducts of aged, nonlaying hens was compared to that in laying hens. The PRs in the mature oviducts of chicks that were diethylstilbestrol (DES) primed and of chicks with DES withdrawn were similarly analyzed for comparative purposes. The oviducts from the DES-withdrawn chicks and from aged nonlaying hens showed marked atrophy. The PR concentrations in the oviducts of DES-withdrawn chicks and of aged nonlaying hens were reduced to one half and one fourth those of controls, respectively. The oviduct atrophy and reduced PR concentrations in the nonlaying hen were not caused by decreased plasma estrogen since estrogen concentrations were similar in aged nonlaying hen and in the laying hens. The stabilities of the PR from laying and nonlaying hen oviducts were identical. However, analysis in vivo and in vitro revealed that the PR from the oviducts of DES-withdrawn chicks and of aged, nonlaying hens had lost the capacity for nuclear translocation and binding. This was accompanied in the DES-withdrawn chick by the inability of progesterone to alter RNA synthesis in the oviduct in vivo. This loss in nuclear binding capacity of PR in both animal models was accompanied by a loss of one of the molecular species of the PR (the B species). The loss of the B species differs from the loss of the A species that occurs during the winter in the domestic fowl. Thus, two types of regulation of the receptor may exist. The results suggest that biologically inactive receptors may explain the cessation of reproductive function in these aged animals.