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Conformational Constraining of Inactive and Active States of a Seven Transmembrane Receptor by Metal Ion Site Engineering in the Extracellular End of Transmembrane Segment V

Laboratory for Molecular Pharmacology, Department of Pharmacology, the Panum Institute; University of Copenhagen, Denmark (M.M.R., I.O., T.B-J., T.W.S); and Institute of Biochemistry, Faculty of Biosciences, Pharmacy and Psychology, University of Leipzig, Leipzig, Germany (R.D., U.G., A.G.B-S).

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, 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 with wild-type ORF74-HHV8 with decreased agonist (CXCL1/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 µM) acted as an allosteric enhancer as it increased the B_max (7.1-fold), the potency (9.9-fold), the affinity (1.7- 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) were 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.

Address correspondence to: Mette M. Rosenkilde, Laboratory for Molecular Pharmacology, Department of Pharmacology, The Panum Institute 18.6, Bleg-damsvej 3, 2200 Copenhagen N, Denmark. E-mail: rosenkilde{at}molpharm.dk.

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