title: Interruption of G protein-coupling in CXCR2 does not alter ligand binding, but eliminates ligand-activation of GTPgamma35S binding, calcium mobilization, and chemotaxis.
abstract: CXCR2 is a seven-transmembrane receptor that transduces intracellular signals in response to the chemokines IL-8, MGSA/GRO, and other ELR motif-containing CXC chemokines by coupling to heterotrimeric GTP-binding proteins. In this study, we have mutated two putative G protein-coupling regions of CXCR2 and characterized the effects of these mutations on ligand-activated signal transductions: aspartic acid 89 in the second transmembrane domain and the HRAMR sequence (BBXXB motif, found in the third intracellular loop where B indicates a basic amino acid and X represents any amino acid). The Asp89 was replaced by either asparagine (D89N) or glutamic acid (D89E). For the BBXXB motif, the first two basic amino acids were mutated to two neutral isoleucines (HR-II), or alternatively, two isoleucines were inserted between alanine and methionine (II-insert). When expressed in human embryonic kidney 293 cells, the D89E mutant was localized intracellularly with no detectable cell surface expression. In contrast, D89N, HR-II, and II-insert mutants displayed cell surface expression, with Kd values and expression levels similar to that of the wild-type transfectant. The ability of the mutants to transduce signal was assessed by ligand-stimulated GTPgamma35S binding, mobilization of intracellular free Ca2+, and chemotaxis assays. Both D89N and HR-II mutants signaled similarly to a wild-type receptor in all three assays. However, the II-insert mutant exhibited a loss of ligand-stimulated GTPgamma35S binding, calcium mobilization, and chemotaxis. Unexpectedly, this receptor underwent ligand-induced sequestration comparable to wild-type CXCR2. These data indicate that Asp89 and the basic amino acids in the third intracellular domain do not play essential roles in ligand-induced signal transduction through CXCR2. However, proper secondary structure and orientation of the third intracellular loop of CXCR2 are essential for ligand-mediated signal transduction but not for receptor sequestration.
authors.affiliation: Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2175, USA.
authors.forename: W
authors.initials: W
authors.lastname: Yang
authors.forename: W P
authors.initials: WP
authors.lastname: Schraw
authors.forename: S G
authors.initials: SG
authors.lastname: Mueller
authors.forename: A
authors.initials: A
authors.lastname: Richmond
mesh_terms: D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D054360:Chemokine CXCL1; D019743:Chemokines, CXC; D002630:Chemotactic Factors / Q000378:metabolism; D002633:Chemotaxis; D004705:Endocytosis; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006133:Growth Substances / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D008024:Ligands; D009154:Mutation; D011485:Protein Binding; D019707:Receptors, Chemokine / Q000235:genetics / Q000378:metabolism*; D018123:Receptors, Interleukin / Q000235:genetics / Q000378:metabolism*; D023063:Receptors, Interleukin-8B; D013462:Sulfur Radioisotopes
chemical_list: C475829:CXCL1 protein, human; D054360:Chemokine CXCL1; D019743:Chemokines, CXC; D002630:Chemotactic Factors; D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; D008024:Ligands; D019707:Receptors, Chemokine; D018123:Receptors, Interleukin; D023063:Receptors, Interleukin-8B; D013462:Sulfur Radioisotopes; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins; D002118:Calcium