Functional differences between human formyl peptide receptor isoforms 26, 98, and G6.
The formyl peptide receptor (FPR) is expressed in neutrophils, couples to G(i)-proteins and activates phospholipase C, chemotaxis and cytotoxic cell functions. FPR isoforms 26, 98, and G6 differ from each other in amino acids 101, 192 and 346 (FPR-26: V101, N192, E346; FPR-98: L101, N192, A346; FPR-G6: V101, K192, A346), but the functional significance of those structural differences is unknown. In order to address this question, we analyzed FPR-26, FPR-98 and FPR-G6 by co-expressing recombinant FLAG epitope-tagged FPRs with the G-protein G(i)alpha(2)beta(1)gamma(2) in Sf9 insect cells and measured high-affinity agonist binding and guanosine 5'- O-(3-thiotriphosphate) (GTPgammaS) binding. The B(max) values of high-affinity agonist binding with FPR-98 and FPR-G6 were much lower than with FPR-26. FPR-98 and FPR-G6 activated considerably fewer G(i)-proteins, and were much less constitutively active, than FPR-26. Whereas FPR-26 migrated as a monomer in SDS polyacrylamide electrophoresis, FPR-98 and FPR-G6 migrated as dimers and tetramers. In terms of immunoreactivity, FRP-98 and FPR-G6 were expressed at higher levels than FPR-26. Single amino acid exchanges at positions 101 (V-->L), 192 (N-->K) and 346 (E-->A) in FPR-26 revealed that E346 accounts for FPR-26 migrating as a monomer and the high constitutive activity of FPR-26. The V101L, N192K and E346A exchanges all reduced high-affinity agonist binding and the number of G(i)-proteins activated by FPR-26. We conclude that (i) FPR isoforms 98 and G6 exhibit a partial G(i)-protein coupling defect relative to FPR-26 and that (ii) E346 critically determines constitutive activity, G(i)-protein coupling and physical state of FPR-26.
Wenzel-Seifert
Katharina
K
Department of Pharmacology and Toxicology, The University of Kansas, Malott Hall, Room 5064, 1251 Wescoe Hall Drive, Lawrence, KS 66045-7582, USA.
Seifert
Roland
R
D000595:Amino Acid Sequence; D002478:Cells, Cultured; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D016297:Mutagenesis, Site-Directed; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D044042:Receptors, Formyl Peptide / Q000235:genetics*; D013460:Sulfur Isotopes; D014316:Tritium
D020033:Protein Isoforms; D044042:Receptors, Formyl Peptide; D013460:Sulfur Isotopes; D014316:Tritium; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D009240:N-Formylmethionine Leucyl-Phenylalanine; D019204:GTP-Binding Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go