Receptor Structural Features Determining Drug Tolerance

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Drug tolerance is a key issue in understanding drug addiction and is of great importance in the treatment of chronic diseases. G-protein coupled receptors (GPCRs) are among the most important cellular targets for illicit drugs and are responsible for the action of approximately 60% of drugs on the market today. Biochemical data have implicated the C-terminus of these receptors with desensitization towards the drug and internalization within the cell. These processes have been implicated with the onset of drug tolerance. It is thought that phosphorylation of the C- terminus of GPCRs and the resulting interaction with beta-arrestin, a cytosolic regulatory protein, governs the fate of these receptors following activation. The precise structural detail as to how these events are mediated is not known. The cannabinoid receptor (CB1) is a GPCR responsible for the biological effects of delta 9-tetrahydrocannabinol, the active constituent of marijuana. This receptor serves as an excellent model for the investigation of structural properties underlying these mechanisms. The biochemical evidence leads to the hypothesis that the C-terminus of CB1 is able to form differing complexes with beta-arrestin, with alternate complexes resulting in desensitization only, internalization only, or both. This project is a pilot study where Nuclear Magnetic Resonance Spectroscopy of carefully designed peptides will be used to address the following specific aims i) to determine a structural model of the C-terminus of CB1 ii) to measure the effects of phosphorylation on structure iii) to determine appropriate conditions and measure kinetics of binding of peptides to beta-arrestin. The long-term goal of this project is to attain precise knowledge of the interaction between arrestin and the receptor and it is a first step towards understanding the molecular details concerning tolerance. This knowledge will benefit human health by providing a basis for the design of ligands that selectively prevent desensitization while allowing internalization, or conversely, prevent internalization while allowing desensitization. Such ligands will be valuable as biological tools in evaluating how these processes contribute to drug addiction. In addition, this information may contribute to the design of therapeutic agents that circumvent tolerance.