Received for publication June 1, 2006.
Revised August 25, 2006.
Accepted for publication September 12, 2006.

Conformational constraining of inactive and active states of a 7TM receptor by metal-ion site engineering in the extracellular end of transmembrane segment V (TM-V)

Abstract

The extracellular part of transmembrane segment V (TM-V) is expected to be involved in the activation process of 7TM receptors but its role is far from clear. Here we study the highly constitutively active CXC-chemokine receptor encoded by human herpesvirus 8 (ORF74-HHV8) in which a metal-ion site was introduced at the extracellular end of TM-V by substitution of two arginines at positions V:01 and V:05 with histidines [R208H;R212H]. The metal-ion site conferred high potency inverse agonist properties (EC₅₀ of 1.7 µM) to Zn(II) in addition to agonist and allosteric enhancing properties at concentrations >10 µM. The chemokine interaction with [R208H;R212H]-ORF74 was altered compared to wild-type ORF74-HHV8 with decreased agonist (CXCL/GRO) potency (84 fold), affinity (5.8 and 136 fold in competition against agonist and inverse agonist, respectively) and binding capacity, B_max (25 fold). Zn(II) in activating concentrations (100 µ) acted as an allosteric enhancer as it increased the B_max (7.1 fold), the potency (9.9 fold), the affinity (1.7 fold and 6.1 fold in competition against agonist and inverse agonist, respectively) and the efficacy (2.5 fold) of CXCL1/GRO. The activating properties of Zn(II) was not due to a metal-ion site between the ligand and the receptor because CXCL1/GRO analogs in which the putative metal-ion binding residues had been substituted - [H19A] and [H34A] - acted like wild-type CXCL1/GRO. Based on the complex action of Zn(II) and on the chemokine interaction for [R208H;R212H]-ORF74 we conclude that the extracellular end of TM-V is important for the activation of this CXC-chemokine receptor.

Key words: Chemotactic peptides, Gq/11 family, IP3/DAG, Func. analysis receptor/ion channel mutants, Mass Spectroscopy, Mutagenesis/Chimeric approaches, Receptor binding studies

This article has been cited by other articles:

				P. C. Jensen, R. Nygaard, S. Thiele, A. Elder, G. Zhu, R. Kolbeck, S. Ghosh, T. W. Schwartz, and M. M. Rosenkilde Molecular Interaction of a Potent Nonpeptide Agonist with the Chemokine Receptor CCR8 Mol. Pharmacol., August 1, 2007; 72(2): 327 - 340. [Abstract] [Full Text] [PDF]