Mutagenesis and computer modeling studies of a GPCR conserved residue W5.43(194) in ligand recognition and signal transduction for CB2 receptor

Zhang, Yuxun; Xie, Zhaojun; Wang, Lirong; Schreiter, Brielle; Lazo, John S; Gertsch, Jurg; Xie, Xiang-Qun (2011). Mutagenesis and computer modeling studies of a GPCR conserved residue W5.43(194) in ligand recognition and signal transduction for CB2 receptor. International immunopharmacology, 11(9), pp. 1303-10. Amsterdam: Elsevier 10.1016/j.intimp.2011.04.013

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W5.43(194), a conserved tryptophan residue among G-protein coupled receptors (GPCRs) and cannabinoid receptors (CB), was examined in the present report for its significance in CB2 receptor ligand binding and adenylyl cyclase (AC) activity. Computer modeling postulates that this site in CB2 may be involved in the affinity of WIN55212-2 and SR144528 through aromatic contacts. In the present study, we reported that a CB2 receptor mutant, W5.43(194)Y, which had a tyrosine (Y) substitution for tryptophan (W), retained the binding affinity for CB agonist CP55940, but reduced binding affinity for CB2 agonist WIN55212-2 and inverse agonist SR144528 by 8-fold and 5-fold, respectively; the CB2 W5.43(194)F and W5.43(194)A mutations significantly affect the binding activities of CP55940, WIN55212-2 and SR144528. Furthermore, we found that agonist-mediated inhibition of the forskolin-induced cAMP production was dramatically diminished in the CB2 mutant W5.43(194)Y, whereas W5.43(194)F and W5.43(194)A mutants resulted in complete elimination of downstream signaling, suggesting that W5.43(194) was essential for the full activation of CB2. These results indicate that both aromatic interaction and hydrogen bonding are involved in ligand binding for the residue W5.43(194), and the mutations of this tryptophan site may affect the conformation of the ligand binding pocket and therefore control the active conformation of the wild type CB2 receptor. W5.43(194)Y/F/A mutations also displayed noticeable enhancement of the constitutive activation probably attributed to the receptor conformational changes resulted from the mutations.

Item Type:	Journal Article (Original Article)
Division/Institute:	04 Faculty of Medicine > Pre-clinic Human Medicine > Institute of Biochemistry and Molecular Medicine
UniBE Contributor:	Gertsch, Jürg
ISSN:	1567-5769
Publisher:	Elsevier
Language:	English
Submitter:	Factscience Import
Date Deposited:	04 Oct 2013 14:23
Last Modified:	05 Dec 2022 14:06
Publisher DOI:	10.1016/j.intimp.2011.04.013
PubMed ID:	21539938
Web of Science ID:	000294877600023
URI:	https://boris.unibe.ch/id/eprint/8160 (FactScience: 213648)