{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,17]],"date-time":"2025-10-17T13:22:58Z","timestamp":1760707378838},"reference-count":51,"publisher":"Wiley","issue":"1-2","license":[{"start":{"date-parts":[[2004,8,31]],"date-time":"2004-08-31T00:00:00Z","timestamp":1093910400000},"content-version":"vor","delay-in-days":3165,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["European Journal of Biochemistry"],"published-print":{"date-parts":[[1996,1]]},"abstract":"<jats:p>The human C5a receptor (C5aR) belongs to the family of G\u2010protein\u2010coupled receptors with seven transmembrane helices. This part of the molecule is thought to contain part of the ligand\u2010binding pocket, specifically to bind the C\u2010terminal Arg of human C5a. Guided by sequence similarity and molecular modelling studies, several residues including polar (Asn119, Thr168, Gln259) as well as all conserved charged amino acids in the upper transmembrane region of the C5aR (Asp37, Asp82, Arg175, Arg206, Asp282) were exchanged by site\u2010directed mutagenesis. Receptor mutants were transiently expressed in COS cells and analyzed for altered binding behaviour and\/or localization at the cell surface by immunofluorescence. For all residues, suitable mutants could be found that exhibited wild\u2010type affinity towards the ligand, providing evidence against a major contribution of these residues to high\u2010affinity ligand binding. Some mutants, however, exhibited a complete (Asp282\u2192Ala) or partial loss of ligand\u2010binding capacity (Arg175\u2192Ala, Arg206\u2192Gln) despite adequate expression levels on the cell surface. This phenotype was further analyzed in the [Gln206]C5aR mutant: quantitative flow cytometric analysis of epitope\u2010tagged receptor derivatives in 293 cells confirmed an equal level of wild\u2010type and mutant C5aR on the cell surface. Competitive binding curves revealed the presence of only a small population (&lt;10%) of high\u2010affinity sites (<jats:italic>K<\/jats:italic><jats:sub>d<\/jats:sub>\u22482nM), which was functionally active at 20 nM in the heterologous <jats:italic>Xenopus<\/jats:italic> oocyte expression system after coexpression of G\u03b1\u201016. The number of high\u2010affinity sites of wild\u2010type and [Gln206]C5aR in 293 cells could be up\u2010regulated by coexpression of Gi\u03b1\u20102 and down\u2010regulated by GTP[\u03b3S]\u2010mediated uncoupling of the G\u2010protein receptor interaction in membrane preparations. These findings are compatible with a model in which the Arg206 residue located in the upper third of transmembrane helix V determines high\u2010affinity binding in the human C5aR by affecting the intracellular G\u2010protein coupling.<\/jats:p>","DOI":"10.1111\/j.1432-1033.1996.00082.x","type":"journal-article","created":{"date-parts":[[2004,8,31]],"date-time":"2004-08-31T15:30:15Z","timestamp":1093966215000},"page":"82-90","source":"Crossref","is-referenced-by-count":38,"title":["Site\u2010Directed Mutagenesis of Conserved Charged Residues in the Helical Region of the Human C5a Receptor"],"prefix":"10.1111","volume":"235","author":[{"given":"Ute","family":"Raffetseder","sequence":"first","affiliation":[]},{"given":"Detlef","family":"R\u00f6per","sequence":"additional","affiliation":[]},{"given":"Laurence","family":"Mery","sequence":"additional","affiliation":[]},{"given":"Claudia","family":"Gietz","sequence":"additional","affiliation":[]},{"given":"Andreas","family":"Klos","sequence":"additional","affiliation":[]},{"given":"Joachim","family":"Gr\u00f6tzinger","sequence":"additional","affiliation":[]},{"given":"Axel","family":"Wollmer","sequence":"additional","affiliation":[]},{"given":"Fran\u00e7ois","family":"Boulay","sequence":"additional","affiliation":[]},{"given":"J\u00f6rg","family":"K\u00f6hl","sequence":"additional","affiliation":[]},{"given":"Wilfried","family":"Bautsch","sequence":"additional","affiliation":[]}],"member":"311","published-online":{"date-parts":[[2004,8,31]]},"reference":[{"key":"e_1_2_2_2_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0171-2985(11)80316-5"},{"key":"e_1_2_2_3_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.90.11.5133"},{"key":"e_1_2_2_4_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi00226a002"},{"key":"e_1_2_2_5_1","doi-asserted-by":"publisher","DOI":"10.1111\/j.1432-1033.1994.tb18571.x"},{"key":"e_1_2_2_6_1","doi-asserted-by":"publisher","DOI":"10.1016\/0014-5793(93)81464-B"},{"key":"e_1_2_2_7_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.270.27.15966"},{"key":"e_1_2_2_8_1","doi-asserted-by":"crossref","first-page":"14622","DOI":"10.1016\/S0021-9258(18)82378-X","article-title":"The fifth transmembrane segment of the neuromedin B receptor is critical for high affinity neuromedin B binding","volume":"268","author":"Fathi Z.","year":"1993","journal-title":"J. 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