The overall objective of the proposals is to gain structural knowledge of the transmembrane domain of the cannabinoid receptor. Understanding the basis of cannabinoid action as well as design of potential therapeutics requires detailed information about interhelical interactions of transmembrane segments within the protein.
The specific aims are to demonstrate the transmembrane segments of the receptor form specific helix-helix interactions, to characterize the nature of those interactions in order to generate structural information, and to reassemble active receptor from fragments to demonstrate the necessity of specific transmembrane segment interactions to receptor function. Transmembrane segments will be expressed as fusion proteins in E. Coli and assayed for their ability to dimerize. Mutations will be designed and made to characterize interactions necessary for dimerization. The data generated will be used to construct a 3-dimensional model of the CB1 transmembrane domain. In order to reassemble active receptor, CB1 will be expressed as pairs of discontinuous fragments that together comprise the full length protein. CB1 reassembly can provide evidence that transmembrane domain of CB1 acts as an organizing center for proper assembly. It is possible that the methods to be employed will be applicable to other membrane proteins, thereby providing the tools necessary to study the structures of other drugs receptors and membrane proteins in general.
