Abstract
The fourth cytoplasmic domain, the so-called C-terminal juxtamembrane segment or helix VIII, has been identified in numerous G-protein-coupled receptors and exhibits unique functional characteristics. Efforts have been devoted to studying the juxtamembrane segment in order to understand the biological importance of the segment in G-protein activation of the cannabinoid CB1 and CB2 receptors. Recent biochemical data revealed that the CB1 C-terminal juxtamembrane peptide fragment CB1-(401-417) can directly activate the G-protein and also showed that the specificity of the signal transduction activation by the C-terminal juxtamembrane region is unique to the CB1 receptor but not to the CB2 receptor (Mukhopadhyay, S., and Howlett, A. C. (2001) Eur. J. Biochem. 268, 499-505). However, there is experimental work, not yet reported, on the conformational analyses and structural comparison between the respective helix VIII segments of the two receptors. In the present study, we have examined the conformational specificities of the cytoplasmic helical domains for both cannabinoid receptors. Three-dimensional structural features of two synthetic CB1 and CB2 peptides, CB1I397-G418 and CB2I298-K319, respectively, in membrane mimetic DPC micelles were studied using a combined high resolution NMR and computer modeling approach. Comparisons of the NMR-determined structures of the two peptides as well as their correspondent mutant peptides revealed their conformational properties and salt bridge dissimilarity, which might help us to understand the different structural roles of the fourth cytoplasmic helices in the function and regulation of CB1 and CB2 receptors.
Highlights
The cannabinoid (CB)1 receptor subtypes, CB1 and CB2, have been cloned and classified into the class A rhodopsin-like family of the seven transmembrane G-protein-coupled receptors (GPCRs) [1, 2]
They concluded that the C-terminal juxtamembrane fragment peptide CB1 401– 417 can directly activate the Gi protein and, in addition, the specificity of the C-terminal juxtamembrane region’s affinity to Gi and Go proteins is unique to the CB1 receptor (19 –21) but not to the CB2 receptor [21]
We have determined the three-dimensional structures of two peptides corresponding to the extended fourth cytoplasmic juxtamembrane domains within the CB1 and CB2 receptors by means of proton NMR measurements and NMR-derived, distance-constrained dynamics simulation
Summary
The cannabinoid (CB) receptor subtypes, CB1 and CB2, have been cloned and classified into the class A rhodopsin-like family of the seven transmembrane G-protein-coupled receptors (GPCRs) [1, 2]. These subtypes have been identified as important drug discovery targets for numerous potential ther-. Feng and Song [22] investigated the role of two cysteine residues in the C-terminal juxtamembrane region of human CB2 with site-directed mutagenesis They found that the C313A and C320A mutations markedly reduced functional coupling to adenylate cyclase but had no effect on ligand binding and agonist-induced receptor desensitization.
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