IDENTIFICATION OF G-PROTEIN BINDING-SITES OF THE HUMAN INTERLEUKIN-8 RECEPTORS BY FUNCTIONAL MAPPING OF THE INTRACELLULAR LOOPS

Interleukin 8 (IL-8) is considered to be a major mediator of the infla mmatory response. Recent evidence indicates that a direct physical ass ociation occurs between IL-8 receptors and the alpha subunit of guanin e nucleotide regulatory protein (G(i) alpha(2)) upon stimulation of hu man neutrophils by IL-8. In the present study, we identified by site-d irected mutagenesis key residues within the three intracellular loops of the IL-8RA receptor involved in the interaction with G(i) alpha(2). We first systematically mutated, in groups of two to four, all the re sidues in the three intracellular loops of the IL-8 type A receptor to alanine and analyzed the mutant receptors transiently expressed in 29 3 cells. Four residues in the second intracellular loop (Y136, L137, I 139, V140) and one residue in the third intracellular loop (M241) were shown to be crucial for mediating calcium signaling in response to IL -8. Other residues in the second and third intracellular loops were al so found to affect IL-8RA-mediated signaling, but to a lesser extent. These effects were not due to lower expression or low IL-8 binding aff inities to the mutated receptors. Mutagenesis of the residues in the f irst intracellular loop had only weak effects on the mobilization of c alcium induced by IL-8. We then used a coimmunoprecipitation protocol with anti-G(i) alpha(2) antibodies to determine the involvement of the two regions defined above in G(i) alpha(2) coupling to IL-8 type A re ceptors. Whereas the anti-G(i) alpha(2) antibodies coimmunoprecipitate d IL-8 receptors in the wild-type cells, this interaction was lost in cells expressing mutated receptors that affected intracellular calcium mobilization. The peptides corresponding to the regions of the type A receptor found to be critical for G(i) alpha(2) coupling and inductio n of intracellular calcium mobilization were next introduced into cell s expressing wild-type IL-8RA or IL-8RB to assess their role in coupli ng G(i) alpha(2) to both IL-8 receptors. The results obtained in the l atter experiments suggest that the same regions of the second intracel lular loop (Y136, L137, I139, V140) and of the third intracellular loo p (M241) are critically involved in the coupling of both IL-8RA and IL -8 RB to G(i) alpha(2) as well as to a downstream effector (or effecte rs) involved in calcium mobilization.