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Functional Coupling of NKR-P1 Receptors to Various Heterotrimeric G Proteins in Rat Interleukin-2-activated Natural Killer Cells

1997; Elsevier BV; Volume: 272; Issue: 50 Linguagem: Inglês

10.1074/jbc.272.50.31604

1083-351X

A al-Aoukaty, Bent Rolstad, Azzam A. Maghazachi,

T-cell and B-cell Immunology

NKR-P1 molecules constitute a family of type II membrane receptors in natural killer (NK) cells that preferentially activate NK cell killing and release of interferon-γ from these cells. Here, we demonstrate that anti-NKR-P1 enhances GTP binding in rat interleukin-2-activated NK cell membranes; GTP binding to Gi3α, Gsα, Gq,11α, and Gzα increased noticeably in these cell membranes after treatment with anti-NKR-P1. Western blot analysis of membrane proteins prepared from interleukin-2-activated NK cells reveals the presence of Gi1,2α, Gi3α, Goα, Gsα, Gq,11α, Gzα, and G12α, but not G13α. However, only αi3, αs, αq,11, and αz, but not αi1,2, αo, α12, or α13 subunits when immunoprecipitated with the appropriate anti-G protein antibodies, are associated with NKR-P1 when immunoblotted with anti-NKR-P1. Reciprocally, NKR-P1 immunoprecipitated with anti-NKR-P1 is associated with αi3, αs, αq,11, and αz immunoblotted with anti-G proteins. These results are the first to demonstrate the physical and functional coupling of NKR-P1 to the heterotrimeric G proteins in NK cells. NKR-P1 molecules constitute a family of type II membrane receptors in natural killer (NK) cells that preferentially activate NK cell killing and release of interferon-γ from these cells. Here, we demonstrate that anti-NKR-P1 enhances GTP binding in rat interleukin-2-activated NK cell membranes; GTP binding to Gi3α, Gsα, Gq,11α, and Gzα increased noticeably in these cell membranes after treatment with anti-NKR-P1. Western blot analysis of membrane proteins prepared from interleukin-2-activated NK cells reveals the presence of Gi1,2α, Gi3α, Goα, Gsα, Gq,11α, Gzα, and G12α, but not G13α. However, only αi3, αs, αq,11, and αz, but not αi1,2, αo, α12, or α13 subunits when immunoprecipitated with the appropriate anti-G protein antibodies, are associated with NKR-P1 when immunoblotted with anti-NKR-P1. Reciprocally, NKR-P1 immunoprecipitated with anti-NKR-P1 is associated with αi3, αs, αq,11, and αz immunoblotted with anti-G proteins. These results are the first to demonstrate the physical and functional coupling of NKR-P1 to the heterotrimeric G proteins in NK cells. Natural killer (NK) 1The abbreviations used are: NK, natural killer; G protein, guanine nucleotide-binding protein; IP3, inositol trisphosphate; NRS, normal rabbit serum; OX8, human CD8 α/α equivalent; PVDF, polyvinylidene difluoride; HRP, horseradish peroxidase; IL, interleukin; PBS, phosphate-buffered saline; mAb, monoclonal antibody; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; ELISA, enzyme-linked immunosorbent assay; TBS, Tris-buffered saline; TTBS, Tris-buffered saline with Tween 20.1The abbreviations used are: NK, natural killer; G protein, guanine nucleotide-binding protein; IP3, inositol trisphosphate; NRS, normal rabbit serum; OX8, human CD8 α/α equivalent; PVDF, polyvinylidene difluoride; HRP, horseradish peroxidase; IL, interleukin; PBS, phosphate-buffered saline; mAb, monoclonal antibody; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; ELISA, enzyme-linked immunosorbent assay; TBS, Tris-buffered saline; TTBS, Tris-buffered saline with Tween 20.cells were first discovered by their ability to kill certain tumor cell lines without prior sensitization, but they can also recognize and destroy virally infected cells (1Trinchieri G. Adv. Immunol. 1989; 47: 187-376Crossref PubMed Scopus (2691) Google Scholar, 2Biron C.A. Su H.C. Orange S.J. Methods: Companion Methods Enzymol. 1996; 9: 379-393Crossref Scopus (38) Google Scholar, 3Moretta L. Minagari M.C. Pende D. Botlino C. Biassoni R. Moretta A. J. Clin. Immunol. 1996; 16: 243-253Crossref PubMed Scopus (34) Google Scholar). These cells recognize the major histocompatibility complex class I molecules on target cells, resulting in either inhibition or activation of their cytolytic potential (4Raulet D.H. Nature. 1992; 385: 21-22Crossref Scopus (32) Google Scholar, 5Karlhofer F.M. Ribaudo R.K. Yokoyama W.M. Nature. 1992; 358: 66-70Crossref PubMed Scopus (726) Google Scholar, 6Lanier L.L. Phillips J.H. Semin. Immunol. 1995; 7: 75-82Crossref PubMed Scopus (35) Google Scholar, 7Kaufman D.S. Schoon R.A. Robertson M.J. Leibson P.J. Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 6484-6488Crossref PubMed Scopus (79) Google Scholar). Target cell recognition by rodent NK cells involves C-type lectin proteins, such as NKR-P1 and Ly 49, that are expressed preferentially on NK cells (8Yokoyama W.M. Seaman W.E. Annu. Rev. Immunol. 1993; 11: 613-635Crossref PubMed Scopus (445) Google Scholar). Three homologous NKR-P1 genes have been identified both in mice and rats and are designated as NKR-P1 (A, B, and C) (9Yokoyama W.M. Ryan J.C. Hunter J.J. Smith H.R.C. Stark M. Seaman W.E. J. Immunol. 1991; 147: 3229-3236PubMed Google Scholar, 10Giorda R. Rudent W.A. Vavassori C. Chambers W.H. Hiserodt J.C. Trucco M. Science. 1990; 249: 1298-1300Crossref PubMed Scopus (220) Google Scholar, 11Giorda R. Trucco M. J. Immunol. 1992; 147: 1701-1708Google Scholar, 12Ryan J.C. Turck J. Niemi E.C. Yokoyama W.M. Seaman W.E. J. Immunol. 1992; 149: 1631-1635PubMed Google Scholar, 13Ryan J.C. Seaman W.E. Immunol. Rev. 1997; 155: 79-89Crossref PubMed Scopus (121) Google Scholar). In human NK cells, NKR-P1A has about 46% homology to the rodent NKR-P1 molecules (14Lanier L.L. Chang C. Phillips J.H. J. Immunol. 1994; 153: 2417-2428PubMed Google Scholar). Anti-NKR-P1 monoclonal antibody (3.2.3) reacts with rat NKR-P1 members and induces the production of IP3, the mobilization of intracellular calcium, the secretion of interferon-γ, and the degranulation and cytotoxicity of NK cells (15Ryan J.C. Niemi E.C. Goldfien R.D. Hiserodt J.C. Seaman W.E. J. Immunol. 1991; 147: 3244-3250PubMed Google Scholar, 16Chambers W.H. Vujanovic N.L. DeLeo A.B. Olszowy M.W. Herberman R.B. Hiserodt J.C. J. Exp. Med. 1989; 169: 1373-1389Crossref PubMed Scopus (382) Google Scholar, 17Hisashi A. Noriko A. Takashi S. J. Exp. Med. 1996; 183: 2391-2396Crossref PubMed Scopus (303) Google Scholar).Recently, we reported that the heterotrimeric guanine nucleotide-binding (G) proteins play important roles in mediating rat NK cell lysis of allogeneic and tumor target cells (18Maghazachi A.A. Al-Aoukaty A. Naper C. Torgersen K.M. Rolstad B. J. Immunol. 1996; 157: 5308-5314PubMed Google Scholar). The heterotrimeric G proteins are composed of three subunits (α, β, and γ). In its inactive form, the α-subunit binds the guanine nucleotide GDP and exchanges it with GTP upon activation. Both the α-GTP and the βγ-heterodimer transduce regulatory signals from a large number of cell-surface receptors to various intracellular enzymes such as adenylyl cyclases, phosphodiesterases, and phospholipases (19Liu M. Simon M.I. Nature. 1996; 382: 83-87Crossref PubMed Scopus (189) Google Scholar,20Litosch I. Biochem. J. 1996; 319: 173-178Crossref PubMed Scopus (11) Google Scholar). The ability of NKR-P1 to induce various biological activities in NK cells suggests that multiple intracellular signaling pathways may be activated upon ligating NKR-P1. The presence of a number of different G proteins in rat NK cell membranes suggests that some of these may also be involved in the transmission of various signals in NK cells. Since it is not known to what extent signal transmission through NKR-P1 triggering is dependent on G proteins, we have investigated the physical and functional coupling of different heterotrimeric G proteins to NKR-P1 in NK cells.DISCUSSIONIn the present study, we demonstrate that the anti-NKR-P1 mAb 3.2.3, which recognizes certain members of the NKR-P1 family of NK cell receptors, enhances the GTP binding in rat IL-2-activated NK cell membranes. NKR-P1 are 60-kDa homodimeric proteins belonging to the family of transmembrane glycoprotein receptors with lectin domains (16Chambers W.H. Vujanovic N.L. DeLeo A.B. Olszowy M.W. Herberman R.B. Hiserodt J.C. J. Exp. Med. 1989; 169: 1373-1389Crossref PubMed Scopus (382) Google Scholar), and were first characterized as activating receptors (15Ryan J.C. Niemi E.C. Goldfien R.D. Hiserodt J.C. Seaman W.E. J. Immunol. 1991; 147: 3244-3250PubMed Google Scholar). Although the natural ligand for NKR-P1 is still undefined, 3.2.3 antibody induces redirected lysis (16Chambers W.H. Vujanovic N.L. DeLeo A.B. Olszowy M.W. Herberman R.B. Hiserodt J.C. J. Exp. Med. 1989; 169: 1373-1389Crossref PubMed Scopus (382) Google Scholar), transduces signals important for regulating NK cell growth (17Hisashi A. Noriko A. Takashi S. J. Exp. Med. 1996; 183: 2391-2396Crossref PubMed Scopus (303) Google Scholar), and induces intracellular calcium mobilization (15Ryan J.C. Niemi E.C. Goldfien R.D. Hiserodt J.C. Seaman W.E. J. Immunol. 1991; 147: 3244-3250PubMed Google Scholar), phosphoinositide turnover (15Ryan J.C. Niemi E.C. Goldfien R.D. Hiserodt J.C. Seaman W.E. J. Immunol. 1991; 147: 3244-3250PubMed Google Scholar), and interferon-γ secretion (17Hisashi A. Noriko A. Takashi S. J. Exp. Med. 1996; 183: 2391-2396Crossref PubMed Scopus (303) Google Scholar).Signals are transmitted intracellularly via one of two identified pathways: the tyrosine kinase receptors pathway or the G protein-coupled receptor pathway. The G protein intracellular signaling pathway, being the older one, became specialized and has been conserved for at least the last 1.2 billion years (25Neer E.J. Schmidt C.J. Nambudripad R. Smith T.F. Nature. 1994; 371: 297-300Crossref PubMed Scopus (1284) Google Scholar). This pathway is important for the activation of various secondary messengers such as phospholipase, in particular phospholipase Cβ (20Litosch I. Biochem. J. 1996; 319: 173-178Crossref PubMed Scopus (11) Google Scholar, 26Exton J.H. Eur. J. Biochem. 1997; 243: 19-20Crossref Scopus (143) Google Scholar), and the mitogen-activated protein kinase pathway (27Lopez-Ilasaca M. Crespo P. Pellici P.G. Gutkind J.S. Wetzker R. Science. 1997; 275: 394-397Crossref PubMed Scopus (627) Google Scholar). Recent work has shown that the βγ-dimer binds and activates the phosphatidylinositol 3-kinase γ-isoform (27Lopez-Ilasaca M. Crespo P. Pellici P.G. Gutkind J.S. Wetzker R. Science. 1997; 275: 394-397Crossref PubMed Scopus (627) Google Scholar, 28Stoyanov B. Volinia S. Hanck T. Rubio I. Loubtchenkov M. Malek D. Stoyanova S. Vanhaesebroeck B. Dhand R. Nurnberg B. Gierschik P. Seedorf K. Hsuan J.S. Waterfield M.D. Wetzker R. Science. 1995; 269: 690-693Crossref PubMed Scopus (638) Google Scholar). In addition, this dimer binds pleckstrin homology domain (29Pitcher J.A. Touhara K. Payne E.S. Leftkowit R.J. Cell. 1995; 83: 821-830Abstract Full Text PDF PubMed Scopus (289) Google Scholar), suggesting the importance of G proteins in mediating various biological activities inside the cells.More than 20 α-subunits and at least 5 β- and 10 γ-subunits have been identified so far (30Fields T.A. Casey P.J. Biochem. J. 1997; 321: 561-571Crossref PubMed Scopus (247) Google Scholar). The α-subunit is divided into four subfamilies. These are (i) αs (stimulatory of adenylyl cyclases), which includes αs and αolf; (ii) αi (inhibitory of adenylyl cyclases), which includes αi1, αi2, αi3, αo, αt1, αt2, αz, and αgust; (iii) αq(activator of phospholipases), which includes αq, α11, α14, and α15/α16; and (iv) α12, which includes α12 and α13. In its resting state, the α-subunit binds GDP and upon ligation of the receptors, conformational changes occur within the receptor α-subunit initiating the activation of G proteins, resulting in the binding of GTP to the α-subunit and its dissociation from the βγ-dimer (30Fields T.A. Casey P.J. Biochem. J. 1997; 321: 561-571Crossref PubMed Scopus (247) Google Scholar, 31Spiegel A.M. Annu. Rev. Physiol. 1996; 58: 143-170Crossref PubMed Scopus (187) Google Scholar). Both the α-subunit and the βγ-dimer can then interact with various regulatory effector molecules (30Fields T.A. Casey P.J. Biochem. J. 1997; 321: 561-571Crossref PubMed Scopus (247) Google Scholar, 31Spiegel A.M. Annu. Rev. Physiol. 1996; 58: 143-170Crossref PubMed Scopus (187) Google Scholar).Several receptors present on NK cells are coupled to G proteins, which mediate various signals inside these cells. These include: (i) NK cell Fc receptors (32Procopio A.D. Paolini R. Vevhio I Frati L. Santoni A. J. Immunol. 1991; 146: 3550-3556PubMed Google Scholar); (ii) receptors present on human NK cells that recognize tumor targets, and are coupled to Gs and Go (22Maghazachi A.A. Al-Aoukaty A. J. Biol. Chem. 1994; 269: 6796-6802Abstract Full Text PDF PubMed Google Scholar), (iii) receptors present on rat NK cells that recognize tumor or allogeneic target cells, and are coupled to Go and Gz (18Maghazachi A.A. Al-Aoukaty A. Naper C. Torgersen K.M. Rolstad B. J. Immunol. 1996; 157: 5308-5314PubMed Google Scholar), (iv) transforming growth factor-β1 receptors present on rat NK cells, and are coupled to Go and Gs (33Maghazachi A.A. Al-Aoukaty A. Int. Immunol. 1993; 5: 825-832Crossref PubMed Scopus (10) Google Scholar), (v) the CXC chemokine IL-8 receptors present on human NK cells, and are coupled to Go(34Sebok K. Woodside D. Al-Aoukaty A. Ho A.D. Gluck S. Maghazachi A.A. J. Immunol. 1993; 150: 1524-1534PubMed Google Scholar), (vi) the CXC chemokine IP-10 receptors present on human IL-2-activated NK cells, and are coupled to Gi, Go, and Gq (35Maghazachi A.A. Skalhegg B.S. Rolstad B. Al-Aoukaty A. FASEB J. 1997; 11: 765-774Crossref PubMed Scopus (91) Google Scholar), (vii) the CXC chemokine SDF-1 receptors present on human NK cells, and are coupled to Go, Gs, and Gq (36Maghazachi A.A. Biochem. Biophys. Res. Commun. 1997; 236: 270-274Crossref PubMed Scopus (43) Google Scholar), (viii) the CC chemokines MCP-1 and RANTES receptors present on human NK cells, and are coupled to Gi, Go, Gs, and Gz(24Al-Aoukaty A. Schall T.J. Maghazachi A.A. Blood. 1996; 87: 4255-4260Crossref PubMed Google Scholar), (ix) the C chemokine lymphotactin receptors present on human NK cells, and are coupled to Gi, Go, and Gq (35Maghazachi A.A. Skalhegg B.S. Rolstad B. Al-Aoukaty A. FASEB J. 1997; 11: 765-774Crossref PubMed Scopus (91) Google Scholar), and (x) exocytosis of NK cells, which involves certain G proteins (37Ting A.T. Schoon R.A. Abraham R.T. Leibson P.J. J. Biol. Chem. 1992; 267: 23957-23962Abstract Full Text PDF PubMed Google Scholar).Our present results demonstrate that the heterotrimeric G proteins in rat IL-2-activated NK cells are activated upon ligating NKR-P1 receptors with anti-NKR-P1 antibody. Utilizing the ELISA and the immunoselection assays with magnetic beads and antibodies specific for various subtypes of G proteins, we were able to determine the binding of GTP to various G protein α-subunits in NK cell membranes. Our results clearly demonstrate that Gi3, Gs, Gq, and Gz, but not Gi1,2, Go, G12, or G13 are activated upon the binding of anti-NKR-P1 antibody to NKR-P1 molecules. Furthermore, we established that there is a physical association of NKR-P1 molecules with these G proteins. This was clearly demonstrated by immunoprecipitating NK cell membranes with antibodies to the α-subunit of Gi3, Gs, Gq, or Gz and then immunoblotting with anti-NKR-P1 antibody and, reciprocally, by immunoprecipitating the membranes with anti-NKR-P1 antibody and then immunoblotting with antibodies to the α-subunits of Gi3, Gs, Gq, or Gz.Although NKR-P1 is a single-transmembrane-spanning domain receptor, and does not belong to the seven-transmembrane-spanning domain receptors, which characteristically bind the heterotrimeric G proteins, other single transmembrane-spanning domain receptors such as transforming growth factor-β1 receptors (33Maghazachi A.A. Al-Aoukaty A. Int. Immunol. 1993; 5: 825-832Crossref PubMed Scopus (10) Google Scholar, 38Kataoka R. Sherlock J. Lanier S.M. J. Biol. Chem. 1993; 268: 19851-19857Abstract Full Text PDF PubMed Google Scholar), or insulin like growth factor-1 receptors (39Davies P. Patton W. J. Cell Physiol. 1994; 159: 399-406Crossref PubMed Scopus (11) Google Scholar) also bind G proteins. It is interesting that both transforming growth factor-β1 type II receptors (40Lin H.Y. Wang X.-F. Ng-Eaton E. Weinberg R. Lodish H. Cell. 1992; 68: 775-785Abstract Full Text PDF PubMed Scopus (966) Google Scholar) and NKR-P1 receptors (10Giorda R. Rudent W.A. Vavassori C. Chambers W.H. Hiserodt J.C. Trucco M. Science. 1990; 249: 1298-1300Crossref PubMed Scopus (220) Google Scholar, 11Giorda R. Trucco M. J. Immunol. 1992; 147: 1701-1708Google Scholar) are rich in serine/threonine kinases. Whether these kinases form a motif in the single-transmembrane-spanning domain receptor that binds G proteins is an intriguing possibility that needs to be examined.In summary, our results are the first to show the functional coupling of NKR-P1, a type II plasma membrane receptor to various heterotrimeric G proteins in NK cell membranes. The promiscuous coupling of four different G proteins in these membranes to NKR-P1 may contribute to our understanding of the diverse biological functions attributed to this family of molecules in NK cells. Natural killer (NK) 1The abbreviations used are: NK, natural killer; G protein, guanine nucleotide-binding protein; IP3, inositol trisphosphate; NRS, normal rabbit serum; OX8, human CD8 α/α equivalent; PVDF, polyvinylidene difluoride; HRP, horseradish peroxidase; IL, interleukin; PBS, phosphate-buffered saline; mAb, monoclonal antibody; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; ELISA, enzyme-linked immunosorbent assay; TBS, Tris-buffered saline; TTBS, Tris-buffered saline with Tween 20.1The abbreviations used are: NK, natural killer; G protein, guanine nucleotide-binding protein; IP3, inositol trisphosphate; NRS, normal rabbit serum; OX8, human CD8 α/α equivalent; PVDF, polyvinylidene difluoride; HRP, horseradish peroxidase; IL, interleukin; PBS, phosphate-buffered saline; mAb, monoclonal antibody; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; ELISA, enzyme-linked immunosorbent assay; TBS, Tris-buffered saline; TTBS, Tris-buffered saline with Tween 20.cells were first discovered by their ability to kill certain tumor cell lines without prior sensitization, but they can also recognize and destroy virally infected cells (1Trinchieri G. Adv. Immunol. 1989; 47: 187-376Crossref PubMed Scopus (2691) Google Scholar, 2Biron C.A. Su H.C. Orange S.J. Methods: Companion Methods Enzymol. 1996; 9: 379-393Crossref Scopus (38) Google Scholar, 3Moretta L. Minagari M.C. Pende D. Botlino C. Biassoni R. Moretta A. J. Clin. Immunol. 1996; 16: 243-253Crossref PubMed Scopus (34) Google Scholar). These cells recognize the major histocompatibility complex class I molecules on target cells, resulting in either inhibition or activation of their cytolytic potential (4Raulet D.H. Nature. 1992; 385: 21-22Crossref Scopus (32) Google Scholar, 5Karlhofer F.M. Ribaudo R.K. Yokoyama W.M. Nature. 1992; 358: 66-70Crossref PubMed Scopus (726) Google Scholar, 6Lanier L.L. Phillips J.H. Semin. Immunol. 1995; 7: 75-82Crossref PubMed Scopus (35) Google Scholar, 7Kaufman D.S. Schoon R.A. Robertson M.J. Leibson P.J. Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 6484-6488Crossref PubMed Scopus (79) Google Scholar). Target cell recognition by rodent NK cells involves C-type lectin proteins, such as NKR-P1 and Ly 49, that are expressed preferentially on NK cells (8Yokoyama W.M. Seaman W.E. Annu. Rev. Immunol. 1993; 11: 613-635Crossref PubMed Scopus (445) Google Scholar). Three homologous NKR-P1 genes have been identified both in mice and rats and are designated as NKR-P1 (A, B, and C) (9Yokoyama W.M. Ryan J.C. Hunter J.J. Smith H.R.C. Stark M. Seaman W.E. J. Immunol. 1991; 147: 3229-3236PubMed Google Scholar, 10Giorda R. Rudent W.A. Vavassori C. Chambers W.H. Hiserodt J.C. Trucco M. Science. 1990; 249: 1298-1300Crossref PubMed Scopus (220) Google Scholar, 11Giorda R. Trucco M. J. Immunol. 1992; 147: 1701-1708Google Scholar, 12Ryan J.C. Turck J. Niemi E.C. Yokoyama W.M. Seaman W.E. J. Immunol. 1992; 149: 1631-1635PubMed Google Scholar, 13Ryan J.C. Seaman W.E. Immunol. Rev. 1997; 155: 79-89Crossref PubMed Scopus (121) Google Scholar). In human NK cells, NKR-P1A has about 46% homology to the rodent NKR-P1 molecules (14Lanier L.L. Chang C. Phillips J.H. J. Immunol. 1994; 153: 2417-2428PubMed Google Scholar). Anti-NKR-P1 monoclonal antibody (3.2.3) reacts with rat NKR-P1 members and induces the production of IP3, the mobilization of intracellular calcium, the secretion of interferon-γ, and the degranulation and cytotoxicity of NK cells (15Ryan J.C. Niemi E.C. Goldfien R.D. Hiserodt J.C. Seaman W.E. J. Immunol. 1991; 147: 3244-3250PubMed Google Scholar, 16Chambers W.H. Vujanovic N.L. DeLeo A.B. Olszowy M.W. Herberman R.B. Hiserodt J.C. J. Exp. Med. 1989; 169: 1373-1389Crossref PubMed Scopus (382) Google Scholar, 17Hisashi A. Noriko A. Takashi S. J. Exp. Med. 1996; 183: 2391-2396Crossref PubMed Scopus (303) Google Scholar). Recently, we reported that the heterotrimeric guanine nucleotide-binding (G) proteins play important roles in mediating rat NK cell lysis of allogeneic and tumor target cells (18Maghazachi A.A. Al-Aoukaty A. Naper C. Torgersen K.M. Rolstad B. J. Immunol. 1996; 157: 5308-5314PubMed Google Scholar). The heterotrimeric G proteins are composed of three subunits (α, β, and γ). In its inactive form, the α-subunit binds the guanine nucleotide GDP and exchanges it with GTP upon activation. Both the α-GTP and the βγ-heterodimer transduce regulatory signals from a large number of cell-surface receptors to various intracellular enzymes such as adenylyl cyclases, phosphodiesterases, and phospholipases (19Liu M. Simon M.I. Nature. 1996; 382: 83-87Crossref PubMed Scopus (189) Google Scholar,20Litosch I. Biochem. J. 1996; 319: 173-178Crossref PubMed Scopus (11) Google Scholar). The ability of NKR-P1 to induce various biological activities in NK cells suggests that multiple intracellular signaling pathways may be activated upon ligating NKR-P1. The presence of a number of different G proteins in rat NK cell membranes suggests that some of these may also be involved in the transmission of various signals in NK cells. Since it is not known to what extent signal transmission through NKR-P1 triggering is dependent on G proteins, we have investigated the physical and functional coupling of different heterotrimeric G proteins to NKR-P1 in NK cells. DISCUSSIONIn the present study, we demonstrate that the anti-NKR-P1 mAb 3.2.3, which recognizes certain members of the NKR-P1 family of NK cell receptors, enhances the GTP binding in rat IL-2-activated NK cell membranes. NKR-P1 are 60-kDa homodimeric proteins belonging to the family of transmembrane glycoprotein receptors with lectin domains (16Chambers W.H. Vujanovic N.L. DeLeo A.B. Olszowy M.W. Herberman R.B. Hiserodt J.C. J. Exp. Med. 1989; 169: 1373-1389Crossref PubMed Scopus (382) Google Scholar), and were first characterized as activating receptors (15Ryan J.C. Niemi E.C. Goldfien R.D. Hiserodt J.C. Seaman W.E. J. Immunol. 1991; 147: 3244-3250PubMed Google Scholar). Although the natural ligand for NKR-P1 is still undefined, 3.2.3 antibody induces redirected lysis (16Chambers W.H. Vujanovic N.L. DeLeo A.B. Olszowy M.W. Herberman R.B. Hiserodt J.C. J. Exp. Med. 1989; 169: 1373-1389Crossref PubMed Scopus (382) Google Scholar), transduces signals important for regulating NK cell growth (17Hisashi A. Noriko A. Takashi S. J. Exp. Med. 1996; 183: 2391-2396Crossref PubMed Scopus (303) Google Scholar), and induces intracellular calcium mobilization (15Ryan J.C. Niemi E.C. Goldfien R.D. Hiserodt J.C. Seaman W.E. J. Immunol. 1991; 147: 3244-3250PubMed Google Scholar), phosphoinositide turnover (15Ryan J.C. Niemi E.C. Goldfien R.D. Hiserodt J.C. Seaman W.E. J. Immunol. 1991; 147: 3244-3250PubMed Google Scholar), and interferon-γ secretion (17Hisashi A. Noriko A. Takashi S. J. Exp. Med. 1996; 183: 2391-2396Crossref PubMed Scopus (303) Google Scholar).Signals are transmitted intracellularly via one of two identified pathways: the tyrosine kinase receptors pathway or the G protein-coupled receptor pathway. The G protein intracellular signaling pathway, being the older one, became specialized and has been conserved for at least the last 1.2 billion years (25Neer E.J. Schmidt C.J. Nambudripad R. Smith T.F. Nature. 1994; 371: 297-300Crossref PubMed Scopus (1284) Google Scholar). This pathway is important for the activation of various secondary messengers such as phospholipase, in particular phospholipase Cβ (20Litosch I. Biochem. J. 1996; 319: 173-178Crossref PubMed Scopus (11) Google Scholar, 26Exton J.H. Eur. J. Biochem. 1997; 243: 19-20Crossref Scopus (143) Google Scholar), and the mitogen-activated protein kinase pathway (27Lopez-Ilasaca M. Crespo P. Pellici P.G. Gutkind J.S. Wetzker R. Science. 1997; 275: 394-397Crossref PubMed Scopus (627) Google Scholar). Recent work has shown that the βγ-dimer binds and activates the phosphatidylinositol 3-kinase γ-isoform (27Lopez-Ilasaca M. Crespo P. Pellici P.G. Gutkind J.S. Wetzker R. Science. 1997; 275: 394-397Crossref PubMed Scopus (627) Google Scholar, 28Stoyanov B. Volinia S. Hanck T. Rubio I. Loubtchenkov M. Malek D. Stoyanova S. Vanhaesebroeck B. Dhand R. Nurnberg B. Gierschik P. Seedorf K. Hsuan J.S. Waterfield M.D. Wetzker R. Science. 1995; 269: 690-693Crossref PubMed Scopus (638) Google Scholar). In addition, this dimer binds pleckstrin homology domain (29Pitcher J.A. Touhara K. Payne E.S. Leftkowit R.J. Cell. 1995; 83: 821-830Abstract Full Text PDF PubMed Scopus (289) Google Scholar), suggesting the importance of G proteins in mediating various biological activities inside the cells.More than 20 α-subunits and at least 5 β- and 10 γ-subunits have been identified so far (30Fields T.A. Casey P.J. Biochem. J. 1997; 321: 561-571Crossref PubMed Scopus (247) Google Scholar). The α-subunit is divided into four subfamilies. These are (i) αs (stimulatory of adenylyl cyclases), which includes αs and αolf; (ii) αi (inhibitory of adenylyl cyclases), which includes αi1, αi2, αi3, αo, αt1, αt2, αz, and αgust; (iii) αq(activator of phospholipases), which includes αq, α11, α14, and α15/α16; and (iv) α12, which includes α12 and α13. In its resting state, the α-subunit binds GDP and upon ligation of the receptors, conformational changes occur within the receptor α-subunit initiating the activation of G proteins, resulting in the binding of GTP to the α-subunit and its dissociation from the βγ-dimer (30Fields T.A. Casey P.J. Biochem. J. 1997; 321: 561-571Crossref PubMed Scopus (247) Google Scholar, 31Spiegel A.M. Annu. Rev. Physiol. 1996; 58: 143-170Crossref PubMed Scopus (187) Google Scholar). Both the α-subunit and the βγ-dimer can then interact with various regulatory effector molecules (30Fields T.A. Casey P.J. Biochem. J. 1997; 321: 561-571Crossref PubMed Scopus (247) Google Scholar, 31Spiegel A.M. Annu. Rev. Physiol. 1996; 58: 143-170Crossref PubMed Scopus (187) Google Scholar).Several receptors present on NK cells are coupled to G proteins, which mediate various signals inside these cells. These include: (i) NK cell Fc receptors (32Procopio A.D. Paolini R. Vevhio I Frati L. Santoni A. J. Immunol. 1991; 146: 3550-3556PubMed Google Scholar); (ii) receptors present on human NK cells that recognize tumor targets, and are coupled to Gs and Go (22Maghazachi A.A. Al-Aoukaty A. J. Biol. Chem. 1994; 269: 6796-6802Abstract Full Text PDF PubMed Google Scholar), (iii) receptors present on rat NK cells that recognize tumor or allogeneic target cells, and are coupled to Go and Gz (18Maghazachi A.A. Al-Aoukaty A. Naper C. Torgersen K.M. Rolstad B. J. Immunol. 1996; 157: 5308-5314PubMed Google Scholar), (iv) transforming growth factor-β1 receptors present on rat NK cells, and are coupled to Go and Gs (33Maghazachi A.A. Al-Aoukaty A. Int. Immunol. 1993; 5: 825-832Crossref PubMed Scopus (10) Google Scholar), (v) the CXC chemokine IL-8 receptors present on human NK cells, and are coupled to Go(34Sebok K. Woodside D. Al-Aoukaty A. Ho A.D. Gluck S. Maghazachi A.A. J. Immunol. 1993; 150: 1524-1534PubMed Google Scholar), (vi) the CXC chemokine IP-10 receptors present on human IL-2-activated NK cells, and are coupled to Gi, Go, and Gq (35Maghazachi A.A. Skalhegg B.S. Rolstad B. Al-Aoukaty A. FASEB J. 1997; 11: 765-774Crossref PubMed Scopus (91) Google Scholar), (vii) the CXC chemokine SDF-1 receptors present on human NK cells, and are coupled to Go, Gs, and Gq (36Maghazachi A.A. Biochem. Biophys. Res. Commun. 1997; 236: 270-274Crossref PubMed Scopus (43) Google Scholar), (viii) the CC chemokines MCP-1 and RANTES receptors present on human NK cells, and are coupled to Gi, Go, Gs, and Gz(24Al-Aoukaty A. Schall T.J. Maghazachi A.A. Blood. 1996; 87: 4255-4260Crossref PubMed Google Scholar), (ix) the C chemokine lymphotactin receptors present on human NK cells, and are coupled to Gi, Go, and Gq (35Maghazachi A.A. Skalhegg B.S. Rolstad B. Al-Aoukaty A. FASEB J. 1997; 11: 765-774Crossref PubMed Scopus (91) Google Scholar), and (x) exocytosis of NK cells, which involves certain G proteins (37Ting A.T. Schoon R.A. Abraham R.T. Leibson P.J. J. Biol. Chem. 1992; 267: 23957-23962Abstract Full Text PDF PubMed Google Scholar).Our present results demonstrate that the heterotrimeric G proteins in rat IL-2-activated NK cells are activated upon ligating NKR-P1 receptors with anti-NKR-P1 antibody. Utilizing the ELISA and the immunoselection assays with magnetic beads and antibodies specific for various subtypes of G proteins, we were able to determine the binding of GTP to various G protein α-subunits in NK cell membranes. Our results clearly demonstrate that Gi3, Gs, Gq, and Gz, but not Gi1,2, Go, G12, or G13 are activated upon the binding of anti-NKR-P1 antibody to NKR-P1 molecules. Furthermore, we established that there is a physical association of NKR-P1 molecules with these G proteins. This was clearly demonstrated by immunoprecipitating NK cell membranes with antibodies to the α-subunit of Gi3, Gs, Gq, or Gz and then immunoblotting with anti-NKR-P1 antibody and, reciprocally, by immunoprecipitating the membranes with anti-NKR-P1 antibody and then immunoblotting with antibodies to the α-subunits of Gi3, Gs, Gq, or Gz.Although NKR-P1 is a single-transmembrane-spanning domain receptor, and does not belong to the seven-transmembrane-spanning domain receptors, which characteristically bind the heterotrimeric G proteins, other single transmembrane-spanning domain receptors such as transforming growth factor-β1 receptors (33Maghazachi A.A. Al-Aoukaty A. Int. Immunol. 1993; 5: 825-832Crossref PubMed Scopus (10) Google Scholar, 38Kataoka R. Sherlock J. Lanier S.M. J. Biol. Chem. 1993; 268: 19851-19857Abstract Full Text PDF PubMed Google Scholar), or insulin like growth factor-1 receptors (39Davies P. Patton W. J. Cell Physiol. 1994; 159: 399-406Crossref PubMed Scopus (11) Google Scholar) also bind G proteins. It is interesting that both transforming growth factor-β1 type II receptors (40Lin H.Y. Wang X.-F. Ng-Eaton E. Weinberg R. Lodish H. Cell. 1992; 68: 775-785Abstract Full Text PDF PubMed Scopus (966) Google Scholar) and NKR-P1 receptors (10Giorda R. Rudent W.A. Vavassori C. Chambers W.H. Hiserodt J.C. Trucco M. Science. 1990; 249: 1298-1300Crossref PubMed Scopus (220) Google Scholar, 11Giorda R. Trucco M. J. Immunol. 1992; 147: 1701-1708Google Scholar) are rich in serine/threonine kinases. Whether these kinases form a motif in the single-transmembrane-spanning domain receptor that binds G proteins is an intriguing possibility that needs to be examined.In summary, our results are the first to show the functional coupling of NKR-P1, a type II plasma membrane receptor to various heterotrimeric G proteins in NK cell membranes. The promiscuous coupling of four different G proteins in these membranes to NKR-P1 may contribute to our understanding of the diverse biological functions attributed to this family of molecules in NK cells. In the present study, we demonstrate that the anti-NKR-P1 mAb 3.2.3, which recognizes certain members of the NKR-P1 family of NK cell receptors, enhances the GTP binding in rat IL-2-activated NK cell membranes. NKR-P1 are 60-kDa homodimeric proteins belonging to the family of transmembrane glycoprotein receptors with lectin domains (16Chambers W.H. Vujanovic N.L. DeLeo A.B. Olszowy M.W. Herberman R.B. Hiserodt J.C. J. Exp. Med. 1989; 169: 1373-1389Crossref PubMed Scopus (382) Google Scholar), and were first characterized as activating receptors (15Ryan J.C. Niemi E.C. Goldfien R.D. Hiserodt J.C. Seaman W.E. J. Immunol. 1991; 147: 3244-3250PubMed Google Scholar). Although the natural ligand for NKR-P1 is still undefined, 3.2.3 antibody induces redirected lysis (16Chambers W.H. Vujanovic N.L. DeLeo A.B. Olszowy M.W. Herberman R.B. Hiserodt J.C. J. Exp. Med. 1989; 169: 1373-1389Crossref PubMed Scopus (382) Google Scholar), transduces signals important for regulating NK cell growth (17Hisashi A. Noriko A. Takashi S. J. Exp. Med. 1996; 183: 2391-2396Crossref PubMed Scopus (303) Google Scholar), and induces intracellular calcium mobilization (15Ryan J.C. Niemi E.C. Goldfien R.D. Hiserodt J.C. Seaman W.E. J. Immunol. 1991; 147: 3244-3250PubMed Google Scholar), phosphoinositide turnover (15Ryan J.C. Niemi E.C. Goldfien R.D. Hiserodt J.C. Seaman W.E. J. Immunol. 1991; 147: 3244-3250PubMed Google Scholar), and interferon-γ secretion (17Hisashi A. Noriko A. Takashi S. J. Exp. Med. 1996; 183: 2391-2396Crossref PubMed Scopus (303) Google Scholar). Signals are transmitted intracellularly via one of two identified pathways: the tyrosine kinase receptors pathway or the G protein-coupled receptor pathway. The G protein intracellular signaling pathway, being the older one, became specialized and has been conserved for at least the last 1.2 billion years (25Neer E.J. Schmidt C.J. Nambudripad R. Smith T.F. Nature. 1994; 371: 297-300Crossref PubMed Scopus (1284) Google Scholar). This pathway is important for the activation of various secondary messengers such as phospholipase, in particular phospholipase Cβ (20Litosch I. Biochem. J. 1996; 319: 173-178Crossref PubMed Scopus (11) Google Scholar, 26Exton J.H. Eur. J. Biochem. 1997; 243: 19-20Crossref Scopus (143) Google Scholar), and the mitogen-activated protein kinase pathway (27Lopez-Ilasaca M. Crespo P. Pellici P.G. Gutkind J.S. Wetzker R. Science. 1997; 275: 394-397Crossref PubMed Scopus (627) Google Scholar). Recent work has shown that the βγ-dimer binds and activates the phosphatidylinositol 3-kinase γ-isoform (27Lopez-Ilasaca M. Crespo P. Pellici P.G. Gutkind J.S. Wetzker R. Science. 1997; 275: 394-397Crossref PubMed Scopus (627) Google Scholar, 28Stoyanov B. Volinia S. Hanck T. Rubio I. Loubtchenkov M. Malek D. Stoyanova S. Vanhaesebroeck B. Dhand R. Nurnberg B. Gierschik P. Seedorf K. Hsuan J.S. Waterfield M.D. Wetzker R. Science. 1995; 269: 690-693Crossref PubMed Scopus (638) Google Scholar). In addition, this dimer binds pleckstrin homology domain (29Pitcher J.A. Touhara K. Payne E.S. Leftkowit R.J. Cell. 1995; 83: 821-830Abstract Full Text PDF PubMed Scopus (289) Google Scholar), suggesting the importance of G proteins in mediating various biological activities inside the cells. More than 20 α-subunits and at least 5 β- and 10 γ-subunits have been identified so far (30Fields T.A. Casey P.J. Biochem. J. 1997; 321: 561-571Crossref PubMed Scopus (247) Google Scholar). The α-subunit is divided into four subfamilies. These are (i) αs (stimulatory of adenylyl cyclases), which includes αs and αolf; (ii) αi (inhibitory of adenylyl cyclases), which includes αi1, αi2, αi3, αo, αt1, αt2, αz, and αgust; (iii) αq(activator of phospholipases), which includes αq, α11, α14, and α15/α16; and (iv) α12, which includes α12 and α13. In its resting state, the α-subunit binds GDP and upon ligation of the receptors, conformational changes occur within the receptor α-subunit initiating the activation of G proteins, resulting in the binding of GTP to the α-subunit and its dissociation from the βγ-dimer (30Fields T.A. Casey P.J. Biochem. J. 1997; 321: 561-571Crossref PubMed Scopus (247) Google Scholar, 31Spiegel A.M. Annu. Rev. Physiol. 1996; 58: 143-170Crossref PubMed Scopus (187) Google Scholar). Both the α-subunit and the βγ-dimer can then interact with various regulatory effector molecules (30Fields T.A. Casey P.J. Biochem. J. 1997; 321: 561-571Crossref PubMed Scopus (247) Google Scholar, 31Spiegel A.M. Annu. Rev. Physiol. 1996; 58: 143-170Crossref PubMed Scopus (187) Google Scholar). Several receptors present on NK cells are coupled to G proteins, which mediate various signals inside these cells. These include: (i) NK cell Fc receptors (32Procopio A.D. Paolini R. Vevhio I Frati L. Santoni A. J. Immunol. 1991; 146: 3550-3556PubMed Google Scholar); (ii) receptors present on human NK cells that recognize tumor targets, and are coupled to Gs and Go (22Maghazachi A.A. Al-Aoukaty A. J. Biol. Chem. 1994; 269: 6796-6802Abstract Full Text PDF PubMed Google Scholar), (iii) receptors present on rat NK cells that recognize tumor or allogeneic target cells, and are coupled to Go and Gz (18Maghazachi A.A. Al-Aoukaty A. Naper C. Torgersen K.M. Rolstad B. J. Immunol. 1996; 157: 5308-5314PubMed Google Scholar), (iv) transforming growth factor-β1 receptors present on rat NK cells, and are coupled to Go and Gs (33Maghazachi A.A. Al-Aoukaty A. Int. Immunol. 1993; 5: 825-832Crossref PubMed Scopus (10) Google Scholar), (v) the CXC chemokine IL-8 receptors present on human NK cells, and are coupled to Go(34Sebok K. Woodside D. Al-Aoukaty A. Ho A.D. Gluck S. Maghazachi A.A. J. Immunol. 1993; 150: 1524-1534PubMed Google Scholar), (vi) the CXC chemokine IP-10 receptors present on human IL-2-activated NK cells, and are coupled to Gi, Go, and Gq (35Maghazachi A.A. Skalhegg B.S. Rolstad B. Al-Aoukaty A. FASEB J. 1997; 11: 765-774Crossref PubMed Scopus (91) Google Scholar), (vii) the CXC chemokine SDF-1 receptors present on human NK cells, and are coupled to Go, Gs, and Gq (36Maghazachi A.A. Biochem. Biophys. Res. Commun. 1997; 236: 270-274Crossref PubMed Scopus (43) Google Scholar), (viii) the CC chemokines MCP-1 and RANTES receptors present on human NK cells, and are coupled to Gi, Go, Gs, and Gz(24Al-Aoukaty A. Schall T.J. Maghazachi A.A. Blood. 1996; 87: 4255-4260Crossref PubMed Google Scholar), (ix) the C chemokine lymphotactin receptors present on human NK cells, and are coupled to Gi, Go, and Gq (35Maghazachi A.A. Skalhegg B.S. Rolstad B. Al-Aoukaty A. FASEB J. 1997; 11: 765-774Crossref PubMed Scopus (91) Google Scholar), and (x) exocytosis of NK cells, which involves certain G proteins (37Ting A.T. Schoon R.A. Abraham R.T. Leibson P.J. J. Biol. Chem. 1992; 267: 23957-23962Abstract Full Text PDF PubMed Google Scholar). Our present results demonstrate that the heterotrimeric G proteins in rat IL-2-activated NK cells are activated upon ligating NKR-P1 receptors with anti-NKR-P1 antibody. Utilizing the ELISA and the immunoselection assays with magnetic beads and antibodies specific for various subtypes of G proteins, we were able to determine the binding of GTP to various G protein α-subunits in NK cell membranes. Our results clearly demonstrate that Gi3, Gs, Gq, and Gz, but not Gi1,2, Go, G12, or G13 are activated upon the binding of anti-NKR-P1 antibody to NKR-P1 molecules. Furthermore, we established that there is a physical association of NKR-P1 molecules with these G proteins. This was clearly demonstrated by immunoprecipitating NK cell membranes with antibodies to the α-subunit of Gi3, Gs, Gq, or Gz and then immunoblotting with anti-NKR-P1 antibody and, reciprocally, by immunoprecipitating the membranes with anti-NKR-P1 antibody and then immunoblotting with antibodies to the α-subunits of Gi3, Gs, Gq, or Gz. Although NKR-P1 is a single-transmembrane-spanning domain receptor, and does not belong to the seven-transmembrane-spanning domain receptors, which characteristically bind the heterotrimeric G proteins, other single transmembrane-spanning domain receptors such as transforming growth factor-β1 receptors (33Maghazachi A.A. Al-Aoukaty A. Int. Immunol. 1993; 5: 825-832Crossref PubMed Scopus (10) Google Scholar, 38Kataoka R. Sherlock J. Lanier S.M. J. Biol. Chem. 1993; 268: 19851-19857Abstract Full Text PDF PubMed Google Scholar), or insulin like growth factor-1 receptors (39Davies P. Patton W. J. Cell Physiol. 1994; 159: 399-406Crossref PubMed Scopus (11) Google Scholar) also bind G proteins. It is interesting that both transforming growth factor-β1 type II receptors (40Lin H.Y. Wang X.-F. Ng-Eaton E. Weinberg R. Lodish H. Cell. 1992; 68: 775-785Abstract Full Text PDF PubMed Scopus (966) Google Scholar) and NKR-P1 receptors (10Giorda R. Rudent W.A. Vavassori C. Chambers W.H. Hiserodt J.C. Trucco M. Science. 1990; 249: 1298-1300Crossref PubMed Scopus (220) Google Scholar, 11Giorda R. Trucco M. J. Immunol. 1992; 147: 1701-1708Google Scholar) are rich in serine/threonine kinases. Whether these kinases form a motif in the single-transmembrane-spanning domain receptor that binds G proteins is an intriguing possibility that needs to be examined. In summary, our results are the first to show the functional coupling of NKR-P1, a type II plasma membrane receptor to various heterotrimeric G proteins in NK cell membranes. The promiscuous coupling of four different G proteins in these membranes to NKR-P1 may contribute to our understanding of the diverse biological functions attributed to this family of molecules in NK cells. We thank Dr. J. Ryan (University of California, San Francisco, CA) for suggestions during the preparation of this study.