Title

Mapping amino acid residues critical for ligand binding and activity of the human cannabinoid receptor one, a G-protein coupled receptor

Date of Completion

January 2002

Keywords

Biology, Molecular|Biology, Neuroscience

Degree

Ph.D.

Abstract

The human cannabinoid receptor one (CB1) is a G-protein coupled receptor found primarily in the central nervous system. A primary goal in the field of cannabinoid receptor research is to understand how the receptor interacts with its ligands and the signal transduction machinery to produce a physiological result. CB1 provides an excellent target for potential therapeutics including appetite suppressants, analgesics, and antiemetics. ^ This thesis describes three studies of CB1 designed to identify key amino acid residues for ligand binding and receptor activity. The enhanced affinity of CB2, the peripheral cannabinoid receptor, for the ligand WIN 55,212-2 relative to CB1 was examined using chimeric receptors. It was determined that the presence of a serine residue in CB2 compared with a glycine residue in CB1 in transmembrane helix (TM) 3 contributes to the increased affinity of CB2 for WIN 55-212-2. A scanning mutagenesis project was designed to identify regions in the extracellular domain of CB1 involved in ligand binding. A series of mutant receptors, each with a dipeptide insertion, were evaluated for binding to CP 55,940 and SR 141716A. It was found that binding to CP 55,940 but not SR 141716A was sensitive to disruption of extracellular loop (EC)1. In contrast, receptors with insertions in EC3 bound both ligands like wild type. Binding to both CP 55,940 and SR 141716A is impacted by mutation at some positions in EC2 but not others. Furthermore, binding is not sensitive to disruption of the distal portion of the amino terminus by mutation but is sensitive to disruption of the region close to TM1. Two amino acids, leucine 207 and threonine 210, within TM3 of CB1 were shown to be critical for a wild-type like pharmacological profile for five ligands. Using agonists and inverse agonists, the pattern of binding affinities obtained for the mutant receptors, L207A and T210I, compared to the wild type, suggests that they have become shifted towards G-protein activation. This suggests that in the wild type these residues are involved in maintaining the receptor in its resting state. ^

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