Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1993 Sep 1;294(Pt 2):581–587. doi: 10.1042/bj2940581

The role of the third intracellular loop of the neutrophil N-formyl peptide receptor in G protein coupling.

E R Prossnitz 1, O Quehenberger 1, C G Cochrane 1, R D Ye 1
PMCID: PMC1134495  PMID: 8373373

Abstract

The G-protein-coupled N-formyl peptide receptor (FPR) contains one of the smallest known third intracellular loops of this class of receptors, consisting of only 15 amino acids. To study the role of this region of the receptor in G protein coupling and signal transduction, we generated a deletion mutant (D3i) in which 10 amino acids of the loop were removed, as well as a series of site-directed mutants containing substitutions of the charged and polar amino acids of this loop. The D3i mutant, expressed at normal levels on the cell surface, displayed a KD for labelled N-formyl-Met-Leu-Phe ([3H]FMLP) of 165 nM. This value compares with a KD for the wild-type FPR of 1.0 nM, or 20 nM in the presence of guanosine 5'-[gamma-thio]triphosphate, which uncouples G proteins from the receptor. These results indicate that D3i contains significant structural defects, beyond the disruption of G protein coupling, that affect ligand binding properties. Ten site-directed mutants generated in the third intracellular loop (T226A, K227E, H229A, K230Q, K235Q, S236A, S236A/S237G, R238G, R241E and S244A) displayed KD values between 0.5 and 1.0 nM, with expression levels between 22% (K227E) and 111% (H229A) of that of wild type receptor. The capacity of the mutants for signal transductions was determined by measuring intracellular Ca2+ mobilization. Eight of the ten mutants displayed EC50 values for FMLP of between 0.07 and 0.9 nM, as compared with 0.12 nM for the wild-type receptor. The two mutants K227E and R238G had EC50 values of 2.7 and 2.9 nM respectively. The increase in EC50 could be accounted for partially by the low levels of receptor expression. All ten mutants gave maximum levels of Ca2+ mobilization similar to that produced by the wild-type FPR. These results contradict the conclusions reached with other G-protein-coupled receptors and indicate that the third intracellular loop of the FPR does not have a critical role in the functional coupling of G proteins.

Full text

PDF
581

Images in this article

583Figure 2
on p.583

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Boulay F., Tardif M., Brouchon L., Vignais P. Synthesis and use of a novel N-formyl peptide derivative to isolate a human N-formyl peptide receptor cDNA. Biochem Biophys Res Commun. 1990 May 16;168(3):1103–1109. doi: 10.1016/0006-291x(90)91143-g. [DOI] [PubMed] [Google Scholar]
  2. Boulay F., Tardif M., Brouchon L., Vignais P. The human N-formylpeptide receptor. Characterization of two cDNA isolates and evidence for a new subfamily of G-protein-coupled receptors. Biochemistry. 1990 Dec 18;29(50):11123–11133. doi: 10.1021/bi00502a016. [DOI] [PubMed] [Google Scholar]
  3. Cotecchia S., Exum S., Caron M. G., Lefkowitz R. J. Regions of the alpha 1-adrenergic receptor involved in coupling to phosphatidylinositol hydrolysis and enhanced sensitivity of biological function. Proc Natl Acad Sci U S A. 1990 Apr;87(8):2896–2900. doi: 10.1073/pnas.87.8.2896. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cotecchia S., Kobilka B. K., Daniel K. W., Nolan R. D., Lapetina E. Y., Caron M. G., Lefkowitz R. J., Regan J. W. Multiple second messenger pathways of alpha-adrenergic receptor subtypes expressed in eukaryotic cells. J Biol Chem. 1990 Jan 5;265(1):63–69. [PubMed] [Google Scholar]
  5. Dalman H. M., Neubig R. R. Two peptides from the alpha 2A-adrenergic receptor alter receptor G protein coupling by distinct mechanisms. J Biol Chem. 1991 Jun 15;266(17):11025–11029. [PubMed] [Google Scholar]
  6. Dixon R. A., Kobilka B. K., Strader D. J., Benovic J. L., Dohlman H. G., Frielle T., Bolanowski M. A., Bennett C. D., Rands E., Diehl R. E. Cloning of the gene and cDNA for mammalian beta-adrenergic receptor and homology with rhodopsin. Nature. 1986 May 1;321(6065):75–79. doi: 10.1038/321075a0. [DOI] [PubMed] [Google Scholar]
  7. Dixon R. A., Sigal I. S., Candelore M. R., Register R. B., Scattergood W., Rands E., Strader C. D. Structural features required for ligand binding to the beta-adrenergic receptor. EMBO J. 1987 Nov;6(11):3269–3275. doi: 10.1002/j.1460-2075.1987.tb02645.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Franke R. R., König B., Sakmar T. P., Khorana H. G., Hofmann K. P. Rhodopsin mutants that bind but fail to activate transducin. Science. 1990 Oct 5;250(4977):123–125. doi: 10.1126/science.2218504. [DOI] [PubMed] [Google Scholar]
  9. Franke R. R., Sakmar T. P., Graham R. M., Khorana H. G. Structure and function in rhodopsin. Studies of the interaction between the rhodopsin cytoplasmic domain and transducin. J Biol Chem. 1992 Jul 25;267(21):14767–14774. [PubMed] [Google Scholar]
  10. Higashijima T., Uzu S., Nakajima T., Ross E. M. Mastoparan, a peptide toxin from wasp venom, mimics receptors by activating GTP-binding regulatory proteins (G proteins). J Biol Chem. 1988 May 15;263(14):6491–6494. [PubMed] [Google Scholar]
  11. Khorana H. G. Rhodopsin, photoreceptor of the rod cell. An emerging pattern for structure and function. J Biol Chem. 1992 Jan 5;267(1):1–4. [PubMed] [Google Scholar]
  12. Kobilka B. K., Frielle T., Collins S., Yang-Feng T., Kobilka T. S., Francke U., Lefkowitz R. J., Caron M. G. An intronless gene encoding a potential member of the family of receptors coupled to guanine nucleotide regulatory proteins. Nature. 1987 Sep 3;329(6134):75–79. doi: 10.1038/329075a0. [DOI] [PubMed] [Google Scholar]
  13. Kobilka B. K., Kobilka T. S., Daniel K., Regan J. W., Caron M. G., Lefkowitz R. J. Chimeric alpha 2-,beta 2-adrenergic receptors: delineation of domains involved in effector coupling and ligand binding specificity. Science. 1988 Jun 3;240(4857):1310–1316. doi: 10.1126/science.2836950. [DOI] [PubMed] [Google Scholar]
  14. Koo C., Lefkowitz R. J., Snyderman R. Guanine nucleotides modulate the binding affinity of the oligopeptide chemoattractant receptor on human polymorphonuclear leukocytes. J Clin Invest. 1983 Sep;72(3):748–753. doi: 10.1172/JCI111045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Koo C., Lefkowitz R. J., Snyderman R. The oligopeptide chemotactic factor receptor on human polymorphonuclear leukocyte membranes exists in two affinity states. Biochem Biophys Res Commun. 1982 May 31;106(2):442–449. doi: 10.1016/0006-291x(82)91130-5. [DOI] [PubMed] [Google Scholar]
  16. Kozak M. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell. 1986 Jan 31;44(2):283–292. doi: 10.1016/0092-8674(86)90762-2. [DOI] [PubMed] [Google Scholar]
  17. König B., Arendt A., McDowell J. H., Kahlert M., Hargrave P. A., Hofmann K. P. Three cytoplasmic loops of rhodopsin interact with transducin. Proc Natl Acad Sci U S A. 1989 Sep;86(18):6878–6882. doi: 10.1073/pnas.86.18.6878. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Munson P. J., Rodbard D. Ligand: a versatile computerized approach for characterization of ligand-binding systems. Anal Biochem. 1980 Sep 1;107(1):220–239. doi: 10.1016/0003-2697(80)90515-1. [DOI] [PubMed] [Google Scholar]
  19. Nathans J., Hogness D. S. Isolation and nucleotide sequence of the gene encoding human rhodopsin. Proc Natl Acad Sci U S A. 1984 Aug;81(15):4851–4855. doi: 10.1073/pnas.81.15.4851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Nishimoto I., Murayama Y., Katada T., Ui M., Ogata E. Possible direct linkage of insulin-like growth factor-II receptor with guanine nucleotide-binding proteins. J Biol Chem. 1989 Aug 25;264(24):14029–14038. [PubMed] [Google Scholar]
  21. O'Dowd B. F., Hnatowich M., Regan J. W., Leader W. M., Caron M. G., Lefkowitz R. J. Site-directed mutagenesis of the cytoplasmic domains of the human beta 2-adrenergic receptor. Localization of regions involved in G protein-receptor coupling. J Biol Chem. 1988 Nov 5;263(31):15985–15992. [PubMed] [Google Scholar]
  22. Okajima F., Katada T., Ui M. Coupling of the guanine nucleotide regulatory protein to chemotactic peptide receptors in neutrophil membranes and its uncoupling by islet-activating protein, pertussis toxin. A possible role of the toxin substrate in Ca2+-mobilizing receptor-mediated signal transduction. J Biol Chem. 1985 Jun 10;260(11):6761–6768. [PubMed] [Google Scholar]
  23. Okamoto T., Katada T., Murayama Y., Ui M., Ogata E., Nishimoto I. A simple structure encodes G protein-activating function of the IGF-II/mannose 6-phosphate receptor. Cell. 1990 Aug 24;62(4):709–717. doi: 10.1016/0092-8674(90)90116-v. [DOI] [PubMed] [Google Scholar]
  24. Okamoto T., Nishimoto I. Detection of G protein-activator regions in M4 subtype muscarinic, cholinergic, and alpha 2-adrenergic receptors based upon characteristics in primary structure. J Biol Chem. 1992 Apr 25;267(12):8342–8346. [PubMed] [Google Scholar]
  25. Omann G. M., Allen R. A., Bokoch G. M., Painter R. G., Traynor A. E., Sklar L. A. Signal transduction and cytoskeletal activation in the neutrophil. Physiol Rev. 1987 Jan;67(1):285–322. doi: 10.1152/physrev.1987.67.1.285. [DOI] [PubMed] [Google Scholar]
  26. Parkos C. A., Cochrane C. G., Schmitt M., Jesaitis A. J. Regulation of the oxidative response of human granulocytes to chemoattractants. No evidence for stimulated traffic of redox enzymes between endo and plasma membranes. J Biol Chem. 1985 Jun 10;260(11):6541–6547. [PubMed] [Google Scholar]
  27. Peralta E. G., Ashkenazi A., Winslow J. W., Smith D. H., Ramachandran J., Capon D. J. Distinct primary structures, ligand-binding properties and tissue-specific expression of four human muscarinic acetylcholine receptors. EMBO J. 1987 Dec 20;6(13):3923–3929. doi: 10.1002/j.1460-2075.1987.tb02733.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Probst W. C., Snyder L. A., Schuster D. I., Brosius J., Sealfon S. C. Sequence alignment of the G-protein coupled receptor superfamily. DNA Cell Biol. 1992 Jan-Feb;11(1):1–20. doi: 10.1089/dna.1992.11.1. [DOI] [PubMed] [Google Scholar]
  29. Prossnitz E. R., Quehenberger O., Cochrane C. G., Ye R. D. Transmembrane signalling by the N-formyl peptide receptor in stably transfected fibroblasts. Biochem Biophys Res Commun. 1991 Aug 30;179(1):471–476. doi: 10.1016/0006-291x(91)91394-r. [DOI] [PubMed] [Google Scholar]
  30. Quehenberger O., Prossnitz E. R., Cochrane C. G., Ye R. D. Absence of G(i) proteins in the Sf9 insect cell. Characterization of the uncoupled recombinant N-formyl peptide receptor. J Biol Chem. 1992 Oct 5;267(28):19757–19760. [PubMed] [Google Scholar]
  31. Raynor R. L., Zheng B., Kuo J. F. Membrane interactions of amphiphilic polypeptides mastoparan, melittin, polymyxin B, and cardiotoxin. Differential inhibition of protein kinase C, Ca2+/calmodulin-dependent protein kinase II and synaptosomal membrane Na,K-ATPase, and Na+ pump and differentiation of HL60 cells. J Biol Chem. 1991 Feb 15;266(5):2753–2758. [PubMed] [Google Scholar]
  32. Savarese T. M., Fraser C. M. In vitro mutagenesis and the search for structure-function relationships among G protein-coupled receptors. Biochem J. 1992 Apr 1;283(Pt 1):1–19. doi: 10.1042/bj2830001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sklar L. A., Finney D. A., Oades Z. G., Jesaitis A. J., Painter R. G., Cochrane C. G. The dynamics of ligand-receptor interactions. Real-time analyses of association, dissociation, and internalization of an N-formyl peptide and its receptors on the human neutrophil. J Biol Chem. 1984 May 10;259(9):5661–5669. [PubMed] [Google Scholar]
  34. Strader C. D., Dixon R. A., Cheung A. H., Candelore M. R., Blake A. D., Sigal I. S. Mutations that uncouple the beta-adrenergic receptor from Gs and increase agonist affinity. J Biol Chem. 1987 Dec 5;262(34):16439–16443. [PubMed] [Google Scholar]
  35. Weiss E. R., Kelleher D. J., Johnson G. L. Mapping sites of interaction between rhodopsin and transducin using rhodopsin antipeptide antibodies. J Biol Chem. 1988 May 5;263(13):6150–6154. [PubMed] [Google Scholar]
  36. Wigler M., Sweet R., Sim G. K., Wold B., Pellicer A., Lacy E., Maniatis T., Silverstein S., Axel R. Transformation of mammalian cells with genes from procaryotes and eucaryotes. Cell. 1979 Apr;16(4):777–785. doi: 10.1016/0092-8674(79)90093-x. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES