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• W2126762193 abstract "The involvement of basic residues of interleukin(IL)-8 receptors in coupling to the Gi and G16 proteins was investigated by using a series of IL-8 receptor mutants. Substitution of the basic amino acids in the third inner loop of the receptor does not alter the abilities of the receptor mutants to activate recombinant Gα16 or phosphoinositide-specific phospholipase C (PLC) β2 expressed in COS-7 cells. However, an IL-8 receptor mutant with double mutations at residues Lys158 and Arg159of the second inner loop loses its abilities to activate Gα16 but retains its ability to activate PLC β2. The activation of PLC β2 by an IL-8 receptor that is sensitive to pertussis toxin has been previously demonstrated to be mediated through Gβγ. Surprisingly, the IL-8 receptor mutants with substitution of Ala for either residue Lys158 or Arg159 can still activate Gα16, which suggests that either of the two basic residues in the second inner loop of the IL-8 receptor is sufficient for Gα16 coupling. The involvement of basic residues of interleukin(IL)-8 receptors in coupling to the Gi and G16 proteins was investigated by using a series of IL-8 receptor mutants. Substitution of the basic amino acids in the third inner loop of the receptor does not alter the abilities of the receptor mutants to activate recombinant Gα16 or phosphoinositide-specific phospholipase C (PLC) β2 expressed in COS-7 cells. However, an IL-8 receptor mutant with double mutations at residues Lys158 and Arg159of the second inner loop loses its abilities to activate Gα16 but retains its ability to activate PLC β2. The activation of PLC β2 by an IL-8 receptor that is sensitive to pertussis toxin has been previously demonstrated to be mediated through Gβγ. Surprisingly, the IL-8 receptor mutants with substitution of Ala for either residue Lys158 or Arg159 can still activate Gα16, which suggests that either of the two basic residues in the second inner loop of the IL-8 receptor is sufficient for Gα16 coupling. Many biologically active molecules transduce their signals through specific cell-surface receptors. Some of the receptors interact with heterotrimeric GTP-binding proteins (G proteins) 1The abbreviations used are: G protein, heterotrimeric GTP-binding protein; IP, inositol phosphate; IL-8, interleukin-8; IL-8R, IL-8 receptor; PLC, phosphoinositide-specific phospholipase C; PTx, pertussis toxin.1The abbreviations used are: G protein, heterotrimeric GTP-binding protein; IP, inositol phosphate; IL-8, interleukin-8; IL-8R, IL-8 receptor; PLC, phosphoinositide-specific phospholipase C; PTx, pertussis toxin. (1Birnbaumer L. Abramowitz J. Brown A.M. Biochim. Biophys. Acta. 1990; 90: 163-224Crossref Scopus (958) Google Scholar, 2Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4681) Google Scholar). Molecular cloning has revealed the existence of genes encoding at least 20 Gα, 5 Gβ, and 12 Gγ subunits in mammals (3Simon M.I. Strathman M.P. Gautum M. Science. 1991; 252: 802-808Crossref PubMed Scopus (1574) Google Scholar). These subunits can form a variety of heterotrimers that serve to connect specific cell surface receptors to a large number of different effectors including at least 4 PLC β isoforms and many adenylyl cyclases, as well as several specific ion channels (1Birnbaumer L. Abramowitz J. Brown A.M. Biochim. Biophys. Acta. 1990; 90: 163-224Crossref Scopus (958) Google Scholar, 2Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4681) Google Scholar, 3Simon M.I. Strathman M.P. Gautum M. Science. 1991; 252: 802-808Crossref PubMed Scopus (1574) Google Scholar). One of the intriguing questions posed by this apparent complexity is how signal transduction circuits are organized so that different kinds of receptors can be connected to effectors through various G proteins and coordinate a variety of responses in a large number of different cells. The specificity of some of the circuits is determined no doubt by developmental regulation of the expression of genes that encode the receptors, G proteins and effectors. In addition, subcellular localization may contribute to the specificity to a certain extent. However, the primary determinant for formation of a specific signal transduction circuit lies in specific protein-protein interactions.Work has been done to understand the molecular basis of the specificity in receptor-G protein interactions (4Hedin K.E. Duerson K. Clapham D.E. Cell. Signalling. 1993; 5: 505-518Crossref PubMed Scopus (96) Google Scholar). Amino acid sequences that are involved in activation of Gαq have been mapped to the third cytoplasmic (inner) loops of the α1B-adrenergic receptor, the m1 muscarinic receptor, and the glutamate receptors by using various chimeras (5Lechleiter J. Hellmiss R. Duerson K. Ennulat D. David N. Clapham D. Peralta E. EMBO J. 1990; 9: 4381-4390Crossref PubMed Scopus (149) Google Scholar, 6Cotecchia S. Ostrowski J. Kjelsberg M.A. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 1633-1639Abstract Full Text PDF PubMed Google Scholar, 7Pin J.-P. Joly C. Heinemann S.F. Bockaert J. EMBO J. 1994; 13: 342-348Crossref PubMed Scopus (164) Google Scholar, 24Scatchard G. Ann. N. Y. Acad. Sci. 1949; 51: 660-672Crossref Scopus (17757) Google Scholar). Although these sequences share no significant amino acid sequence homology, they appear to be different from the sequences involved in activating Gαs (8Wong S.K.-F. Parker E.M. Ross E.M. J. Biol. Chem. 1990; 265: 6219-6224Abstract Full Text PDF PubMed Google Scholar, 9Kosugi S. Okajima F. Ban T. Hidaka A. Shenker A. Kohn L.D. J. Biol. Chem. 1992; 267: 24153-24156Abstract Full Text PDF PubMed Google Scholar). Recently, we have found that different α1B-adrenergic receptor sequences are involved in coupling to different α subunits of the Gq class (10Wu D. Jiang H. Simon M.I. J. Biol. Chem. 1995; 270: 9828-9832Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Furthermore, receptor sequences in other inner loops have also been implicated in the involvement of G protein coupling. Studies using receptor-derived peptides have implicated that the second inner loop of the N-formyl peptide receptor may be involved in G protein interaction (11Schreiber R.E. Prossnitz E.R. Ye R.D. Cohrane C.G. Bokoch G.M. J. Biol. Chem. 1994; 269: 326-331Abstract Full Text PDF PubMed Google Scholar, 12Bommakanti R.K. Dratz E.A. Siemsen D.W. Jesaitis A.J. Biochemistry. 1995; 34: 6720-6728Crossref PubMed Scopus (35) Google Scholar).We have previously demonstrated that the IL-8 receptor (IL-8R), like many other chemoattractant receptors including the C5a and formyl-methionyl-leucyl-phenylalanine receptors, can couple to both G16 and Gi proteins (14Wu D. LaRosa G.J. Simon M.I. Science. 1993; 261: 101-103Crossref PubMed Scopus (332) Google Scholar). In this report, we will report our investigation of the IL-8R sequences involved in coupling to G16 but not to Gi by site-directed mutagenesis. Our results indicate that two basic amino acid residues in the second inner loop of the IL-8R are essential for coupling to Gα16 but not to Gi, whereas the basic residues in the third inner loop are not required for coupling to either Gi or G16.RESULTS AND DISCUSSIONThe IL-8 receptors were previously shown to couple to two G proteins, Gi and G16 (14Wu D. LaRosa G.J. Simon M.I. Science. 1993; 261: 101-103Crossref PubMed Scopus (332) Google Scholar). To investigate whether different receptor sequences are involved in coupling to these two G proteins, we have generated a series of mutated receptors as tabulated in Fig.1. Since it was postulated that theBBXXB (B stands for basic amino acid, andX stands for any amino acid) motif might be responsible for Gi coupling (15Okamato T. Katada T. Ogata E. Nishimoto I. Cell. 1990; 62: 709-717Abstract Full Text PDF PubMed Scopus (221) Google Scholar), we first investigated whether the BBBXXBmotif (residues Lys247 to Arg251) in the third intracellular loop of the human type B IL-8 receptor is involved in Gi coupling. We constructed the IL-8 receptor mutants, m1, m2, and m3, by substitution of Ala residues for the amino acids Lys246, His247, and Arg248, respectively. These mutants were tested for their abilities to couple to Gi and G16 in a previously established transient transfection assay (10Wu D. Jiang H. Simon M.I. J. Biol. Chem. 1995; 270: 9828-9832Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 14Wu D. LaRosa G.J. Simon M.I. Science. 1993; 261: 101-103Crossref PubMed Scopus (332) Google Scholar, 16Wu D. Katz A. Lee C.-H. Simon M.I. J. Biol. Chem. 1992; 267: 25798-25802Abstract Full Text PDF PubMed Google Scholar, 17Wu D. Katz A. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5297-5301Crossref PubMed Scopus (171) Google Scholar, 18Wu D. Kuang Y. Wu Y. Jiang H. J. Biol. Chem. 1995; 270: 16008-16010Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 19Jiang H. Kuang Y. Wu Y. Smrcka A. Simon M.I. Wu D. J. Biol. Chem. 1996; 271: 13430-13434Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) to characterize the G protein-coupling specificity for the IL-8 receptors. The COS-7 cells used in the assay system do not contain endogenous IL-8 receptors, PLC β2, or Gα16, although they contain Gi2 and PLC β1 (13Wu D. Lee C.H. Rhee S.G. Simon M.I. J. Biol. Chem. 1992; 267: 1811-1817Abstract Full Text PDF PubMed Google Scholar, 14Wu D. LaRosa G.J. Simon M.I. Science. 1993; 261: 101-103Crossref PubMed Scopus (332) Google Scholar, 17Wu D. Katz A. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5297-5301Crossref PubMed Scopus (171) Google Scholar, 20Katz A. Wu D. Simon M.I. Nature. 1992; 360: 686-689Crossref PubMed Scopus (413) Google Scholar). Thus, IL-8 did not elicit any significant elevation of IP levels in cells expressing the IL-8 receptor and its mutants in the absence of Gα16 or PLC β2 (Fig.2 A). To test the G16 coupling of the IL-8 receptor mutants, we cotransfected COS-7 cells with cDNAs encoding Gα16 and the IL-8 receptor or its mutants, and IL-8-induced accumulation of IPs was determined. As shown in Fig.2 B, IL-8 induced marked PTx-resistant accumulation of IPs in cells coexpressing Gα16 and the IL-8 receptor or its mutants, m1, m2, or m3, which suggests that these three IL-8 receptor mutants, like the wild-type IL-8 receptor, can still couple to Gα16. To test the Gi coupling, we cotransfected COS-7 cells with the cDNAs encoding PLC β2 and the receptors. The IL-8 receptor was previously shown to couple to endogenous Gi proteins of COS-7 cells to release Gβγ, which then activates recombinant PLC β2 (14Wu D. LaRosa G.J. Simon M.I. Science. 1993; 261: 101-103Crossref PubMed Scopus (332) Google Scholar). As shown in Fig.2 C, there was clear IL-8-induced accumulation of IPs in cells coexpressing PLC β2 and the IL-8 receptor, m1, m2, or m3, and the ligand-induced responses were mostly PTx-sensitive. Therefore, these data indicate that the IL-8 receptor mutants can couple to both G16 and Gi in transfected COS-7 cells. To test further the importance of the triple basic amino acids in the third inner loop of the IL-8 receptor, these basic amino acids (Lys246-His247-Arg248) were mutated to three alanine residues. As shown in Fig. 2, B andC, the IL-8R mutant can still couple to recombinant Gα16 and to PLC β2 via endogenous Gi proteins. Thus, it is clear that theBBBXXB (residues Lys247 to Arg251) motif at the N-terminal end of the third intracellular loop of the IL-8 receptor is by no means involved in the Gi coupling or the G16 coupling.Another basic amino acid residue in the third inner loop, Lys240, was also investigated for its involvement in coupling to G16 or Gi. We constructed the mutant m5 by substitution of an Ala residue for the residue Lys240. The mutant m5 was subjected to the same tests as m1–4. The tests showed that m5, like the others, can couple to G16 and Gi. Thus, we conclude that the basic residues inside the third inner loop of the human type B IL-8 receptor are not involved in coupling to G16 or Gi.Search of the IL-8 receptor sequence revealed a BBXXXB(Lys158-Lys163) motif in the second inner loop of the receptor. To test whether the basic residue doublet (Lys158-Arg159) is involved in the G protein coupling, we replaced the doublet with two Ala residues creating the mutant m8 (Fig. 1). By testing the mutant in the same cotransfection assay, we found that m8 can induce IP accumulation only in cells coexpressing PLC β2 (Fig.3 B) but not in those coexpressing Gα16 (Fig. 3 A), which suggests that m8 can couple only to Gi but not to Gα16. Neither m6 nor m7, which have substitution of an Ala residue for one of the basic residue doublets, loses its ability to couple to Gα16 (Fig. 3). The ability of m8 to activate PLC β2 has eliminated the possibility that the mutations in m8 greatly changed the conformation of the receptor. Nevertheless, we also did the ligand-binding assay with 125I-IL-8. The expression level of m8 and its affinity for IL-8 are similar to those of the wild-type IL-8 receptor, m6 and m7 (Fig. 1). In addition, we also determined the expression levels of Gα16 in cells coexpressing m8, m6, m7 and the wild-type IL-8 receptor. No major differences were noticed (Fig. 3 C). Therefore, it is reasonable to conclude that either of the basic residues (Lys158 and Arg159) is apparently sufficient to retain the ability of the receptor to couple to G16 and that the presence of either of them is essential for the G16 coupling, although these two residues do not appear to play a significant role in the Gi coupling.Figure 3Effects of mutations in the second inner loop of IL-8R on G protein coupling. COS-7 cells were cotransfected with cDNA encoding β-galactosidase (LacZ), the wild-type IL-8 receptor (IL-8R) or its mutants (m6–8), and cDNA encoding Gα16 (panels Aand C) or PLC β2 (panel B). The cells were treated with (dashed lines) or without (solid lines) 500 ng/ml PTx for 4 h. Then, the levels of IPs in COS-7 cells were determined 20 min after addition of IL-8 (10 n m). The data are presented as means ± S.D., and IL-8-induced accumulation of IPs in cells expressing the wild-type IL-8R was taken as 100% (panel A). The basal level (in the absence of ligand) is about 2300 dpm, and the ligand induced an increase of 4900 dpm in cells expressing the wild-type receptor and Gα16. Extracts from mock transfected cells (lane 1) and cells expressing IL-8R (lane 2), m6 (lane 3), m7 (lane 4), and m8 (lane 5) were also analyzed by Western blotting with a Gα16-specific antibody (panel C).View Large Image Figure ViewerDownload Hi-res image Download (PPT)We have previously demonstrated that different α1-adrenergic receptor sequences are involved in coupling to Gαq/11 and Gα14. However, sequences involved in Gα16 coupling have not been elucidated. Recent reports (18Wu D. Kuang Y. Wu Y. Jiang H. J. Biol. Chem. 1995; 270: 16008-16010Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 21Offermanns S. Simon M.I. J. Biol. Chem. 1995; 270: 15175-15180Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar) shows that Gα16 appears to be promiscuous in its coupling to various receptors. Almost all of the G protein-coupled receptors thus far tested, including Gq-, Gi-, and Gs-coupling receptors, can couple to Gα16 in transfected COS-7 cells (18Wu D. Kuang Y. Wu Y. Jiang H. J. Biol. Chem. 1995; 270: 16008-16010Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 21Offermanns S. Simon M.I. J. Biol. Chem. 1995; 270: 15175-15180Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar). This coupling promiscuity suggests that most G protein-coupling receptors possess the sequence elements and/or conformation required for interaction with and activation of Gα16. Our results provide an insight into what the requirements are. The basic residues Lys158 and Arg159 may constitute the sequence that interacts with and/or activates Gα16 or may be critical for formation of the receptor conformation required for coupling with Gα16. More studies (knowledge of the three-dimensional structure of the receptor) are needed to understand exactly how these two basic residues are involved in Gα16 coupling. Our data also indicate that the BBXXB motif in the third loop of IL-8R is not essential for either Gαi or Gα16 coupling. These data are consistent with the observation that residue Met241 in the third loop, as well as other non-charged amino acid residues in the second loop of IL-8R, are involved in coupling to Gαi2 (22Damaj B.B. McColl S.R. Neote K. Ogborn K.T. Hebert C.A. Naccache P.H. FASEB J. 1996; 10: 1426-1434Crossref PubMed Scopus (57) Google Scholar).Receptor consensus sequences for G protein-coupling were being pursued vigorously in the past. No such sequences have, however, been identified. Therefore, it is now generally believed that each individual receptor possesses specific receptor coupling elements, which were mostly found in the third inner loops of various receptors. Gα16 is an intriguing subunit. It lacks receptor coupling specificity; it couples to various G protein-coupling receptors ranging from Gs to Gi and Gq-coupling receptors. We have been looking for the receptor elements that are required for Gα16 coupling in both α1B-adrenergic receptors (10Wu D. Jiang H. Simon M.I. J. Biol. Chem. 1995; 270: 9828-9832Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), but these elements have been eluding us until we identified the dual basic amino acids in the second loop of the IL-8 receptor. Although we did not identify consensus sequences for G16 coupling, our results are of great significance. 1) These results unequivocally prove that the second loop is involved in G protein-coupling specificity in contrast to most other studies, which usually only implicate the third inner loops. 2) This is the first time that Gα16-coupling elements have been identified. 3) The element required for Gα16 coupling is not required for Gαi coupling. 4) The basic residues in the second and third inner loops, which have been widely believed to be involved in Gi coupling, are not important for Gi coupling by the IL-8 receptor. Therefore, this work provides us with a better understanding of the specific interactions between receptors and G proteins. In addition, the receptor mutants that show limited yet defined G protein-coupling specificity would be useful in determining the specific in vivo function of signal transduction pathways mediated by specific receptors and G proteins. Many biologically active molecules transduce their signals through specific cell-surface receptors. Some of the receptors interact with heterotrimeric GTP-binding proteins (G proteins) 1The abbreviations used are: G protein, heterotrimeric GTP-binding protein; IP, inositol phosphate; IL-8, interleukin-8; IL-8R, IL-8 receptor; PLC, phosphoinositide-specific phospholipase C; PTx, pertussis toxin.1The abbreviations used are: G protein, heterotrimeric GTP-binding protein; IP, inositol phosphate; IL-8, interleukin-8; IL-8R, IL-8 receptor; PLC, phosphoinositide-specific phospholipase C; PTx, pertussis toxin. (1Birnbaumer L. Abramowitz J. Brown A.M. Biochim. Biophys. Acta. 1990; 90: 163-224Crossref Scopus (958) Google Scholar, 2Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4681) Google Scholar). Molecular cloning has revealed the existence of genes encoding at least 20 Gα, 5 Gβ, and 12 Gγ subunits in mammals (3Simon M.I. Strathman M.P. Gautum M. Science. 1991; 252: 802-808Crossref PubMed Scopus (1574) Google Scholar). These subunits can form a variety of heterotrimers that serve to connect specific cell surface receptors to a large number of different effectors including at least 4 PLC β isoforms and many adenylyl cyclases, as well as several specific ion channels (1Birnbaumer L. Abramowitz J. Brown A.M. Biochim. Biophys. Acta. 1990; 90: 163-224Crossref Scopus (958) Google Scholar, 2Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4681) Google Scholar, 3Simon M.I. Strathman M.P. Gautum M. Science. 1991; 252: 802-808Crossref PubMed Scopus (1574) Google Scholar). One of the intriguing questions posed by this apparent complexity is how signal transduction circuits are organized so that different kinds of receptors can be connected to effectors through various G proteins and coordinate a variety of responses in a large number of different cells. The specificity of some of the circuits is determined no doubt by developmental regulation of the expression of genes that encode the receptors, G proteins and effectors. In addition, subcellular localization may contribute to the specificity to a certain extent. However, the primary determinant for formation of a specific signal transduction circuit lies in specific protein-protein interactions. Work has been done to understand the molecular basis of the specificity in receptor-G protein interactions (4Hedin K.E. Duerson K. Clapham D.E. Cell. Signalling. 1993; 5: 505-518Crossref PubMed Scopus (96) Google Scholar). Amino acid sequences that are involved in activation of Gαq have been mapped to the third cytoplasmic (inner) loops of the α1B-adrenergic receptor, the m1 muscarinic receptor, and the glutamate receptors by using various chimeras (5Lechleiter J. Hellmiss R. Duerson K. Ennulat D. David N. Clapham D. Peralta E. EMBO J. 1990; 9: 4381-4390Crossref PubMed Scopus (149) Google Scholar, 6Cotecchia S. Ostrowski J. Kjelsberg M.A. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 1633-1639Abstract Full Text PDF PubMed Google Scholar, 7Pin J.-P. Joly C. Heinemann S.F. Bockaert J. EMBO J. 1994; 13: 342-348Crossref PubMed Scopus (164) Google Scholar, 24Scatchard G. Ann. N. Y. Acad. Sci. 1949; 51: 660-672Crossref Scopus (17757) Google Scholar). Although these sequences share no significant amino acid sequence homology, they appear to be different from the sequences involved in activating Gαs (8Wong S.K.-F. Parker E.M. Ross E.M. J. Biol. Chem. 1990; 265: 6219-6224Abstract Full Text PDF PubMed Google Scholar, 9Kosugi S. Okajima F. Ban T. Hidaka A. Shenker A. Kohn L.D. J. Biol. Chem. 1992; 267: 24153-24156Abstract Full Text PDF PubMed Google Scholar). Recently, we have found that different α1B-adrenergic receptor sequences are involved in coupling to different α subunits of the Gq class (10Wu D. Jiang H. Simon M.I. J. Biol. Chem. 1995; 270: 9828-9832Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Furthermore, receptor sequences in other inner loops have also been implicated in the involvement of G protein coupling. Studies using receptor-derived peptides have implicated that the second inner loop of the N-formyl peptide receptor may be involved in G protein interaction (11Schreiber R.E. Prossnitz E.R. Ye R.D. Cohrane C.G. Bokoch G.M. J. Biol. Chem. 1994; 269: 326-331Abstract Full Text PDF PubMed Google Scholar, 12Bommakanti R.K. Dratz E.A. Siemsen D.W. Jesaitis A.J. Biochemistry. 1995; 34: 6720-6728Crossref PubMed Scopus (35) Google Scholar). We have previously demonstrated that the IL-8 receptor (IL-8R), like many other chemoattractant receptors including the C5a and formyl-methionyl-leucyl-phenylalanine receptors, can couple to both G16 and Gi proteins (14Wu D. LaRosa G.J. Simon M.I. Science. 1993; 261: 101-103Crossref PubMed Scopus (332) Google Scholar). In this report, we will report our investigation of the IL-8R sequences involved in coupling to G16 but not to Gi by site-directed mutagenesis. Our results indicate that two basic amino acid residues in the second inner loop of the IL-8R are essential for coupling to Gα16 but not to Gi, whereas the basic residues in the third inner loop are not required for coupling to either Gi or G16. RESULTS AND DISCUSSIONThe IL-8 receptors were previously shown to couple to two G proteins, Gi and G16 (14Wu D. LaRosa G.J. Simon M.I. Science. 1993; 261: 101-103Crossref PubMed Scopus (332) Google Scholar). To investigate whether different receptor sequences are involved in coupling to these two G proteins, we have generated a series of mutated receptors as tabulated in Fig.1. Since it was postulated that theBBXXB (B stands for basic amino acid, andX stands for any amino acid) motif might be responsible for Gi coupling (15Okamato T. Katada T. Ogata E. Nishimoto I. Cell. 1990; 62: 709-717Abstract Full Text PDF PubMed Scopus (221) Google Scholar), we first investigated whether the BBBXXBmotif (residues Lys247 to Arg251) in the third intracellular loop of the human type B IL-8 receptor is involved in Gi coupling. We constructed the IL-8 receptor mutants, m1, m2, and m3, by substitution of Ala residues for the amino acids Lys246, His247, and Arg248, respectively. These mutants were tested for their abilities to couple to Gi and G16 in a previously established transient transfection assay (10Wu D. Jiang H. Simon M.I. J. Biol. Chem. 1995; 270: 9828-9832Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 14Wu D. LaRosa G.J. Simon M.I. Science. 1993; 261: 101-103Crossref PubMed Scopus (332) Google Scholar, 16Wu D. Katz A. Lee C.-H. Simon M.I. J. Biol. Chem. 1992; 267: 25798-25802Abstract Full Text PDF PubMed Google Scholar, 17Wu D. Katz A. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5297-5301Crossref PubMed Scopus (171) Google Scholar, 18Wu D. Kuang Y. Wu Y. Jiang H. J. Biol. Chem. 1995; 270: 16008-16010Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 19Jiang H. Kuang Y. Wu Y. Smrcka A. Simon M.I. Wu D. J. Biol. Chem. 1996; 271: 13430-13434Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) to characterize the G protein-coupling specificity for the IL-8 receptors. The COS-7 cells used in the assay system do not contain endogenous IL-8 receptors, PLC β2, or Gα16, although they contain Gi2 and PLC β1 (13Wu D. Lee C.H. Rhee S.G. Simon M.I. J. Biol. Chem. 1992; 267: 1811-1817Abstract Full Text PDF PubMed Google Scholar, 14Wu D. LaRosa G.J. Simon M.I. Science. 1993; 261: 101-103Crossref PubMed Scopus (332) Google Scholar, 17Wu D. Katz A. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5297-5301Crossref PubMed Scopus (171) Google Scholar, 20Katz A. Wu D. Simon M.I. Nature. 1992; 360: 686-689Crossref PubMed Scopus (413) Google Scholar). Thus, IL-8 did not elicit any significant elevation of IP levels in cells expressing the IL-8 receptor and its mutants in the absence of Gα16 or PLC β2 (Fig.2 A). To test the G16 coupling of the IL-8 receptor mutants, we cotransfected COS-7 cells with cDNAs encoding Gα16 and the IL-8 receptor or its mutants, and IL-8-induced accumulation of IPs was determined. As shown in Fig.2 B, IL-8 induced marked PTx-resistant accumulation of IPs in cells coexpressing Gα16 and the IL-8 receptor or its mutants, m1, m2, or m3, which suggests that these three IL-8 receptor mutants, like the wild-type IL-8 receptor, can still couple to Gα16. To test the Gi coupling, we cotransfected COS-7 cells with the cDNAs encoding PLC β2 and the receptors. The IL-8 receptor was previously shown to couple to endogenous Gi proteins of COS-7 cells to release Gβγ, which then activates recombinant PLC β2 (14Wu D. LaRosa G.J. Simon M.I. Science. 1993; 261: 101-103Crossref PubMed Scopus (332) Google Scholar). As shown in Fig.2 C, there was clear IL-8-induced accumulation of IPs in cells coexpressing PLC β2 and the IL-8 receptor, m1, m2, or m3, and the ligand-induced responses were mostly PTx-sensitive. Therefore, these data indicate that the IL-8 receptor mutants can couple to both G16 and Gi in transfected COS-7 cells. To test further the importance of the triple basic amino acids in the third inner loop of the IL-8 receptor, these basic amino acids (Lys246-His247-Arg248) were mutated to three alanine residues. As shown in Fig. 2, B andC, the IL-8R mutant can still couple to recombinant Gα16 and to PLC β2 via endogenous Gi proteins. Thus, it is clear that theBBBXXB (residues Lys247 to Arg251) motif at the N-terminal end of the third intracellular loop of the IL-8 receptor is by no means involved in the Gi coupling or the G16 coupling.Another basic amino acid residue in the third inner loop, Lys240, was also investigated for its involvement in coupling to G16 or Gi. We constructed the mutant m5 by substitution of an Ala residue for the residue Lys240. The mutant m5 was subjected to the same tests as m1–4. The tests showed that m5, like the others, can couple to G16 and Gi. Thus, we conclude that the basic residues inside the third inner loop of the human type B IL-8 receptor are not involved in coupling to G16 or Gi.Search of the IL-8 receptor sequence revealed a BBXXXB(Lys158-Lys163) motif in the second inner loop of the receptor. To test whether the basic residue doublet (Lys158-Arg159) is involved in the G protein coupling, we replaced the doublet with two Ala residues creating the mutant m8 (Fig. 1). By testing the mutant in the same cotransfection assay, we found that m8 can induce IP accumulation only in cells coexpressing PLC β2 (Fig.3 B) but not in those coexpressing Gα16 (Fig. 3 A), which suggests that m8 can couple only to Gi but not to Gα16. Neither m6 nor m7, which have substitution of an Ala residue for one of the basic residue doublets, loses its ability to couple to Gα16 (Fig. 3). The ability of m8 to activate PLC β2 has eliminated the possibility that the mutations in m8 greatly changed the conformation of the receptor. Nevertheless, we also did the ligand-binding assay with 125I-IL-8. The expression level of m8 and its affinity for IL-8 are similar to those of the wild-type IL-8 receptor, m6 and m7 (Fig. 1). In addition, we also determined the expression levels of Gα16 in cells coexpressing m8, m6, m7 and the wild-type IL-8 receptor. No major differences were noticed (Fig. 3 C). Therefore, it is reasonable to conclude that either of the basic residues (Lys158 and Arg159) is apparently sufficient to retain the ability of the receptor to couple to G16 and that the presence of either of them is essential for the G16 coupling, although these two residues do not appear to play a significant role in the Gi coupling.We have previously demonstrated that different α1-adrenergic receptor sequences are involved in coupling to Gαq/11 and Gα14. However, sequences involved in Gα16 coupling have not been elucidated. Recent reports (18Wu D. Kuang Y. Wu Y. Jiang H. J. Biol. Chem. 1995; 270: 16008-16010Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 21Offermanns S. Simon M.I. J. Biol. Chem. 1995; 270: 15175-15180Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar) shows that Gα16 appears to be promiscuous in its coupling to various receptors. Almost all of the G protein-coupled receptors thus far tested, including Gq-, Gi-, and Gs-coupling receptors, can couple to Gα16 in transfected COS-7 cells (18Wu D. Kuang Y. Wu Y. Jiang H. J. Biol. Chem. 1995; 270: 16008-16010Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 21Offermanns S. Simon M.I. J. Biol. Chem. 1995; 270: 15175-15180Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar). This coupling promiscuity suggests that most G protein-coupling receptors possess the sequence elements and/or conformation required for interaction with and activation of Gα16. Our results provide an insight into what the requirements are. The basic residues Lys158 and Arg159 may constitute the sequence that interacts with and/or activates Gα16 or may be critical for formation of the receptor conformation required for coupling with Gα16. More studies (knowledge of the three-dimensional structure of the receptor) are needed to understand exactly how these two basic residues are involved in Gα16 coupling. Our data also indicate that the BBXXB motif in the third loop of IL-8R is not essential for either Gαi or Gα16 coupling. These data are consistent with the observation that residue Met241 in the third loop, as well as other non-charged amino acid residues in the second loop of IL-8R, are involved in coupling to Gαi2 (22Damaj B.B. McColl S.R. Neote K. Ogborn K.T. Hebert C.A. Naccache P.H. FASEB J. 1996; 10: 1426-1434Crossref PubMed Scopus (57) Google Scholar).Receptor consensus sequences for G protein-coupling were being pursued vigorously in the past. No such sequences have, however, been identified. Therefore, it is now generally believed that each individual receptor possesses specific receptor coupling elements, which were mostly found in the third inner loops of various receptors. Gα16 is an intriguing subunit. It lacks receptor coupling specificity; it couples to various G protein-coupling receptors ranging from Gs to Gi and Gq-coupling receptors. We have been looking for the receptor elements that are required for Gα16 coupling in both α1B-adrenergic receptors (10Wu D. Jiang H. Simon M.I. J. Biol. Chem. 1995; 270: 9828-9832Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), but these elements have been eluding us until we identified the dual basic amino acids in the second loop of the IL-8 receptor. Although we did not identify consensus sequences for G16 coupling, our results are of great significance. 1) These results unequivocally prove that the second loop is involved in G protein-coupling specificity in contrast to most other studies, which usually only implicate the third inner loops. 2) This is the first time that Gα16-coupling elements have been identified. 3) The element required for Gα16 coupling is not required for Gαi coupling. 4) The basic residues in the second and third inner loops, which have been widely believed to be involved in Gi coupling, are not important for Gi coupling by the IL-8 receptor. Therefore, this work provides us with a better understanding of the specific interactions between receptors and G proteins. In addition, the receptor mutants that show limited yet defined G protein-coupling specificity would be useful in determining the specific in vivo function of signal transduction pathways mediated by specific receptors and G proteins. The IL-8 receptors were previously shown to couple to two G proteins, Gi and G16 (14Wu D. LaRosa G.J. Simon M.I. Science. 1993; 261: 101-103Crossref PubMed Scopus (332) Google Scholar). To investigate whether different receptor sequences are involved in coupling to these two G proteins, we have generated a series of mutated receptors as tabulated in Fig.1. Since it was postulated that theBBXXB (B stands for basic amino acid, andX stands for any amino acid) motif might be responsible for Gi coupling (15Okamato T. Katada T. Ogata E. Nishimoto I. Cell. 1990; 62: 709-717Abstract Full Text PDF PubMed Scopus (221) Google Scholar), we first investigated whether the BBBXXBmotif (residues Lys247 to Arg251) in the third intracellular loop of the human type B IL-8 receptor is involved in Gi coupling. We constructed the IL-8 receptor mutants, m1, m2, and m3, by substitution of Ala residues for the amino acids Lys246, His247, and Arg248, respectively. These mutants were tested for their abilities to couple to Gi and G16 in a previously established transient transfection assay (10Wu D. Jiang H. Simon M.I. J. Biol. Chem. 1995; 270: 9828-9832Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 14Wu D. LaRosa G.J. Simon M.I. Science. 1993; 261: 101-103Crossref PubMed Scopus (332) Google Scholar, 16Wu D. Katz A. Lee C.-H. Simon M.I. J. Biol. Chem. 1992; 267: 25798-25802Abstract Full Text PDF PubMed Google Scholar, 17Wu D. Katz A. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5297-5301Crossref PubMed Scopus (171) Google Scholar, 18Wu D. Kuang Y. Wu Y. Jiang H. J. Biol. Chem. 1995; 270: 16008-16010Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 19Jiang H. Kuang Y. Wu Y. Smrcka A. Simon M.I. Wu D. J. Biol. Chem. 1996; 271: 13430-13434Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) to characterize the G protein-coupling specificity for the IL-8 receptors. The COS-7 cells used in the assay system do not contain endogenous IL-8 receptors, PLC β2, or Gα16, although they contain Gi2 and PLC β1 (13Wu D. Lee C.H. Rhee S.G. Simon M.I. J. Biol. Chem. 1992; 267: 1811-1817Abstract Full Text PDF PubMed Google Scholar, 14Wu D. LaRosa G.J. Simon M.I. Science. 1993; 261: 101-103Crossref PubMed Scopus (332) Google Scholar, 17Wu D. Katz A. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5297-5301Crossref PubMed Scopus (171) Google Scholar, 20Katz A. Wu D. Simon M.I. Nature. 1992; 360: 686-689Crossref PubMed Scopus (413) Google Scholar). Thus, IL-8 did not elicit any significant elevation of IP levels in cells expressing the IL-8 receptor and its mutants in the absence of Gα16 or PLC β2 (Fig.2 A). To test the G16 coupling of the IL-8 receptor mutants, we cotransfected COS-7 cells with cDNAs encoding Gα16 and the IL-8 receptor or its mutants, and IL-8-induced accumulation of IPs was determined. As shown in Fig.2 B, IL-8 induced marked PTx-resistant accumulation of IPs in cells coexpressing Gα16 and the IL-8 receptor or its mutants, m1, m2, or m3, which suggests that these three IL-8 receptor mutants, like the wild-type IL-8 receptor, can still couple to Gα16. To test the Gi coupling, we cotransfected COS-7 cells with the cDNAs encoding PLC β2 and the receptors. The IL-8 receptor was previously shown to couple to endogenous Gi proteins of COS-7 cells to release Gβγ, which then activates recombinant PLC β2 (14Wu D. LaRosa G.J. Simon M.I. Science. 1993; 261: 101-103Crossref PubMed Scopus (332) Google Scholar). As shown in Fig.2 C, there was clear IL-8-induced accumulation of IPs in cells coexpressing PLC β2 and the IL-8 receptor, m1, m2, or m3, and the ligand-induced responses were mostly PTx-sensitive. Therefore, these data indicate that the IL-8 receptor mutants can couple to both G16 and Gi in transfected COS-7 cells. To test further the importance of the triple basic amino acids in the third inner loop of the IL-8 receptor, these basic amino acids (Lys246-His247-Arg248) were mutated to three alanine residues. As shown in Fig. 2, B andC, the IL-8R mutant can still couple to recombinant Gα16 and to PLC β2 via endogenous Gi proteins. Thus, it is clear that theBBBXXB (residues Lys247 to Arg251) motif at the N-terminal end of the third intracellular loop of the IL-8 receptor is by no means involved in the Gi coupling or the G16 coupling. Another basic amino acid residue in the third inner loop, Lys240, was also investigated for its involvement in coupling to G16 or Gi. We constructed the mutant m5 by substitution of an Ala residue for the residue Lys240. The mutant m5 was subjected to the same tests as m1–4. The tests showed that m5, like the others, can couple to G16 and Gi. Thus, we conclude that the basic residues inside the third inner loop of the human type B IL-8 receptor are not involved in coupling to G16 or Gi. Search of the IL-8 receptor sequence revealed a BBXXXB(Lys158-Lys163) motif in the second inner loop of the receptor. To test whether the basic residue doublet (Lys158-Arg159) is involved in the G protein coupling, we replaced the doublet with two Ala residues creating the mutant m8 (Fig. 1). By testing the mutant in the same cotransfection assay, we found that m8 can induce IP accumulation only in cells coexpressing PLC β2 (Fig.3 B) but not in those coexpressing Gα16 (Fig. 3 A), which suggests that m8 can couple only to Gi but not to Gα16. Neither m6 nor m7, which have substitution of an Ala residue for one of the basic residue doublets, loses its ability to couple to Gα16 (Fig. 3). The ability of m8 to activate PLC β2 has eliminated the possibility that the mutations in m8 greatly changed the conformation of the receptor. Nevertheless, we also did the ligand-binding assay with 125I-IL-8. The expression level of m8 and its affinity for IL-8 are similar to those of the wild-type IL-8 receptor, m6 and m7 (Fig. 1). In addition, we also determined the expression levels of Gα16 in cells coexpressing m8, m6, m7 and the wild-type IL-8 receptor. No major differences were noticed (Fig. 3 C). Therefore, it is reasonable to conclude that either of the basic residues (Lys158 and Arg159) is apparently sufficient to retain the ability of the receptor to couple to G16 and that the presence of either of them is essential for the G16 coupling, although these two residues do not appear to play a significant role in the Gi coupling. We have previously demonstrated that different α1-adrenergic receptor sequences are involved in coupling to Gαq/11 and Gα14. However, sequences involved in Gα16 coupling have not been elucidated. Recent reports (18Wu D. Kuang Y. Wu Y. Jiang H. J. Biol. Chem. 1995; 270: 16008-16010Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 21Offermanns S. Simon M.I. J. Biol. Chem. 1995; 270: 15175-15180Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar) shows that Gα16 appears to be promiscuous in its coupling to various receptors. Almost all of the G protein-coupled receptors thus far tested, including Gq-, Gi-, and Gs-coupling receptors, can couple to Gα16 in transfected COS-7 cells (18Wu D. Kuang Y. Wu Y. Jiang H. J. Biol. Chem. 1995; 270: 16008-16010Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 21Offermanns S. Simon M.I. J. Biol. Chem. 1995; 270: 15175-15180Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar). This coupling promiscuity suggests that most G protein-coupling receptors possess the sequence elements and/or conformation required for interaction with and activation of Gα16. Our results provide an insight into what the requirements are. The basic residues Lys158 and Arg159 may constitute the sequence that interacts with and/or activates Gα16 or may be critical for formation of the receptor conformation required for coupling with Gα16. More studies (knowledge of the three-dimensional structure of the receptor) are needed to understand exactly how these two basic residues are involved in Gα16 coupling. Our data also indicate that the BBXXB motif in the third loop of IL-8R is not essential for either Gαi or Gα16 coupling. These data are consistent with the observation that residue Met241 in the third loop, as well as other non-charged amino acid residues in the second loop of IL-8R, are involved in coupling to Gαi2 (22Damaj B.B. McColl S.R. Neote K. Ogborn K.T. Hebert C.A. Naccache P.H. FASEB J. 1996; 10: 1426-1434Crossref PubMed Scopus (57) Google Scholar). Receptor consensus sequences for G protein-coupling were being pursued vigorously in the past. No such sequences have, however, been identified. Therefore, it is now generally believed that each individual receptor possesses specific receptor coupling elements, which were mostly found in the third inner loops of various receptors. Gα16 is an intriguing subunit. It lacks receptor coupling specificity; it couples to various G protein-coupling receptors ranging from Gs to Gi and Gq-coupling receptors. We have been looking for the receptor elements that are required for Gα16 coupling in both α1B-adrenergic receptors (10Wu D. Jiang H. Simon M.I. J. Biol. Chem. 1995; 270: 9828-9832Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), but these elements have been eluding us until we identified the dual basic amino acids in the second loop of the IL-8 receptor. Although we did not identify consensus sequences for G16 coupling, our results are of great significance. 1) These results unequivocally prove that the second loop is involved in G protein-coupling specificity in contrast to most other studies, which usually only implicate the third inner loops. 2) This is the first time that Gα16-coupling elements have been identified. 3) The element required for Gα16 coupling is not required for Gαi coupling. 4) The basic residues in the second and third inner loops, which have been widely believed to be involved in Gi coupling, are not important for Gi coupling by the IL-8 receptor. Therefore, this work provides us with a better understanding of the specific interactions between receptors and G proteins. In addition, the receptor mutants that show limited yet defined G protein-coupling specificity would be useful in determining the specific in vivo function of signal transduction pathways mediated by specific receptors and G proteins. We thank Mark Betz for reading this manuscript." @default.
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• W2126762193 title "Two Basic Amino Acids in the Second Inner Loop of the Interleukin-8 Receptor Are Essential for Gα16 Coupling" @default.
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