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 (EC(50), 1.7 microM) to Zn(II) in addition to agonist and allosteric enhancing properties at concentrations >10 microM. The chemokine interaction with [R208H;R212H]-ORF74 was altered compared with wild-type ORF74-HHV8 with decreased agonist (CXCL1/GROalpha) 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 microM) 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/GROalpha. The activating properties of Zn(II) were not due to a metal ion site between the ligand and the receptor because CXCL1/GROalpha analogs in which the putative metal-ion binding residues had been substituted-[H19A] and [H34A]-acted like wild-type CXCL1/GROalpha. 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.