Functional Coupling of NKR-P1 Receptors to Various Heterotrimeric G Proteins in Rat Interleukin-2-activated Natural Killer 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) 1 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 (1)(2)(3). These cells recognize the major histocompatibility complex class I molecules on target cells, resulting in either inhibition or activation of their cytolytic potential (4 -7). 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 (8). Three homologous NKR-P1 genes have been identified both in mice and rats and are designated as NKR-P1 (A, B, and C) (9 -13). In human NK cells, NKR-P1A has about 46% homology to the rodent NKR-P1 molecules (14). Anti-NKR-P1 monoclonal antibody (3.2.3) reacts with rat NKR-P1 members and induces the production of IP 3 , the mobilization of intracellular calcium, the secretion of interferon-␥, and the degranulation and cytotoxicity of NK cells (15)(16)(17).
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 (18). The heterotrimeric G proteins are composed of three subunits (␣, ␤, and ␥). In its inactive form, the ␣-subunit binds the guanine nucleotide GDP and exchanges it with GTP upon activation. Both the ␣-GTP and the ␤␥-heterodimer transduce regulatory signals from a large number of cell-surface receptors to various intracellular enzymes such as adenylyl cyclases, phosphodiesterases, and phospholipases (19,20). 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.

EXPERIMENTAL PROCEDURES
Animals-Breeding pairs from the rat strains of PVG were bred in our laboratory or were purchased from Harlan Olac Ltd. (Bichester, United Kingdom (UK)).
Isolation and Culturing of NKR-P1 ϩ IL-2-activated NK Cells-This was done according to the method described previously (21). Briefly, rat mononuclear splenocytes were obtained by density gradient centrifugation on Lymphoprep for 30 min at 400 ϫ g, 1.077 g/ml (Nycomed Pharma, Oslo, Norway). The cells were washed and were depleted of CD3 ϩ cells using anti-CD3 monoclonal antibody (G4.18) and rabbit complement. Following incubation for 75 min at 37°C with gentle agitation, the cells were washed several times and incubated with M450 sheep anti-mouse IgG 1 magnetic Dynabeads (Dynal, Oslo, Norway), precoated with mouse anti-rat NKR-P1 mAb (3.2.3), to positively select * This work was supported by grants from the Research Council of Norway and the Norwegian Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § Senior scientist of the Norwegian Cancer Society. 1 The abbreviations used are: NK, natural killer; G protein, guanine nucleotide-binding protein; IP 3 , inositol trisphosphate; NRS, normal rabbit serum; OX8, human CD8 ␣/␣ 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.
Membrane Preparation-This was performed according to our described procedure (22). Briefly, IL-2-activated NK cells were harvested after 7-10 days in culture, washed extensively in ice-cold PBS, and centrifuged at 450 ϫ g for 10 min at 4°C. The cells were suspended in ice-cold lysis buffer containing 10 mM HEPES, pH 7.5, 3 mM MgCl 2 , 2 mM EDTA in addition to the enzyme inhibitors (40 g/ml phenylmethylsulfonyl fluoride, 2 g/ml pepstatin A, 10 g/ml leupeptin, and 2 g/ml aprotinin). After two steps of homogenization and sonication, the mixture was centrifuged at 1000 ϫ g for 10 min at 4°C. The supernatants were transferred to Beckman tubes and ultracetrifuged at 150,000 ϫ g for 45 min at 4°C. The pellets were suspended in a lysis buffer, snap-frozen, and stored at Ϫ80°C.
GTP Binding Assay-GTP binding was measured using a method described by us (23,24). About 50 -100 g of membrane proteins were incubated in 100 l buffer containing 20 mM HEPES/NaOH, pH 7.4, 100 M EDTA, 125 M MgCl 2 , and 10 nM [␥-35 S]GTP (1000 Ci/mmol). The mixture was incubated at 37°C after addition of the indicated antibodies. The reactions were terminated by the addition of 900 l of ice-cold buffer containing 100 mM Tris-HCl, pH 8.0, 25 mM MgCl 2 , 100 mM NaCl, and 20 M unlabeled GTP. The mixtures were incubated on ice for 1 h, washed several times with ice-cold PBS plus 0.05% Tween 20, and centrifuged at 14,000 rpm at 4°C using an Eppendorf centrifuge; the pellets were then suspended in a scintillation mixture and counted in a ␤ counter. Nonspecific binding was determined by the addition of unlabeled GTP.
In addition, an ELISA assay was developed to determine the GTP binding to the ␣-subunit of G proteins. Nunc-Immuno MaxiSorp 96-well plate (removable wells) were coated with goat anti-rabbit IgG for 2 h at 4°C. Rabbit antibodies to various G protein ␣-subtypes, and as a control NRS were added to the plates for additional 2 h. IL-2-activated NK cell membranes stimulated with 10 g/ml anti-NKR-P1 for 1.5 min at 37°C were preincubated in a binding buffer containing 10 nM [␥-35 S]GTP, and were added to these plates. The plates were left on ice for 2 h and then washed three times with ice-cold PBS buffer plus 0.05% Tween 20. Each well was removed and placed in a scintillation vial filled with liquid scintillation mixture and counted in a ␤ counter.
To confirm the nature of G protein ␣-subtypes activated after ligating NKR-P1 with anti-NKR-P1, a method was developed using immunomagnetic beads (Dynabeads) coated with sheep anti-rabbit IgG (Dynal, Oslo, Norway). The beads were incubated for 2 h at 4°C with rabbit anti-G proteins, rabbit IgG, or NRS in a PBS buffer containing 1% bovine serum albumin. IL-2-activated NK cell membranes were incubated first with 10 g/ml anti-NKR-P1 for 1.5 min, added to the GTP binding buffer plus [␥-35 S] GTP, then mixed with anti-G protein-or NRS-coated beads, washed with PBS buffer plus 0.05% Tween 20, suspended in the scintillation mixture and transferred to scintillation vials. All assays were performed in triplicate.
Immunoblot Analysis-Immunoblotting was performed as described (18). Briefly, 100 g of membrane proteins were suspended in SDS sample buffer and separated by 12% SDS-polyacrylamide gel electrophoresis. The proteins were electrotransferred to PVDF membrane, blocked with 5% skim milk in TBS buffer and incubated with proper primary antibody overnight at room temperature, washed twice with TBS plus 0.05% Tween 20 (TTBS), incubated with HRP-conjugated secondary antibody, washed twice with TTBS, and then developed using HRP development reagents (Bio-Rad).
Immunoprecipitation Assay-Membrane pellets were suspended in a solubilization buffer containing 25 mM sodium phosphate, pH 7.4, 5 mM EDTA, 5 mM EGTA, 200 mM KCl, 25% glycerol, and 25 mM MgCl 2 , plus 1% CHAPS. They were centrifuged at 100,000 ϫ g, and the supernatants were collected and stored at Ϫ80°C until the time of the assay. The membranes were added to the solubilization buffer plus 0.3% CHAPS and incubated overnight with rabbit antibodies to various ␣-subtypes of G proteins, rabbit IgG, or NRS at 4°C with gentle agitation. They were mixed with protein A/G-agarose and incubated for additional 4 h. The immunocomplexes were isolated by centrifugation at 14,000 rpm at 4°C using an Eppendorf centrifuge and washed three times with the solubilization buffer plus 0.3% CHAPS. The pellets were suspended in SDS sample buffer boiled for 5 min, and the agarose beads were removed by spinning the tubes at 2000 rpm for 2 min. The immunoprecipitates were separated by 12% SDS-polyacrylamide gel electrophoresis and immunoblotted using monoclonal anti-NKR-P1 primary antibody and goat anti-mouse IgG HRP-conjugated secondary antibody. Similarly, solubilized membranes were immunoprecipitated with anti-NKR-P1 or mouse IgG, and then immunoblotted with rabbit antisera to various G protein ␣-subtypes or with anti-NKR-P1. Goat anti-mouse or goat anti-rabbit-HRP was used as a secondary antibody.
Statistics-Significant values were determined using a two-tailed Student's t test.

RESULTS
Anti-NKR-P1 Enhances the Binding of GTP to IL-2-activated NK Cells-Because the ligand for NKR-P1 is not known, we have used the 3.2.3 mAb directed against this receptor and shown to activate NK cells upon binding (15). Fig. 1A shows that incubating IL-2-activated NK cell membranes with 10 g/ml anti-NKR-P1 (3.2.3 antibody) resulted in a maximum binding of [␥-35 S]GTP (p Ͻ 0.01 when compared with basal binding, or binding to membranes activated with a control mouse IgG antibody). Kinetic studies show that incubating IL-2-activated NK cell membranes with anti-NKR-P1 for 1.5 min gave a maximal GTP binding response (p Ͻ 0.02, Fig. 1B). In contrast, mouse monoclonal antibody to OX8 did not induce GTP binding in NK cell membranes (Fig. 1C). These experiments demonstrate that activation of G proteins with anti-NKR-P1 is a result of a specific interaction of this antibody with NKR-P1 molecules and is not a result of cross-reaction with other unrelated proteins that are abundant in NK cell membranes such as OX8, which is present on more than 50% of NKR-P1 ϩ IL-2-activated NK cells (21). Additionally, mouse IgG, which was used as a control, failed to enhance the GTP binding in these membranes (Fig. 1C).
Immunoselection Method Determines the Coupling of G Protein ␣-Subtypes to NKR-P1 Molecules-To confirm the specific binding results of GTP to various G protein ␣-subtypes in anti-NKR-P1 stimulated NK cell membranes, we developed a method to immunoselect the ␣-subtypes of G proteins with Dynabeads coated with various anti-G protein antibodies. In this method, rabbit anti-G proteins were coated on sheep antirabbit coupled beads. Membranes from NK cells stimulated with anti-NKR-P1 (10 g/ml for 1.5 min at 37°C) were incubated with this mixture. Similar to the ELISA assay, anti-NKR-P1 enhances the GTP binding to ␣ i3 , ␣ s , ␣ q,11 , and ␣ z in NK cell membranes (p Ͻ 0.01, 0.001, 0.001, and 0.005, respectively, when compared with the basal binding), but not to other ␣-subunits of G proteins, as shown in Fig. 2B.
Identification of Various G Protein ␣-Subtypes in IL-2-activated NK Cell Membranes-To determine whether the Dynabeads immunoselection method is also appropriate for the detection of various ␣-subtypes of G proteins present in NK cell membranes, these membranes were incubated with anti-G protein-coupled or IgG-coupled Dynabeads. The membranes were then isolated and immunoblotted with anti-G protein antibodies. ␣ i1,2 , ␣ i3 , ␣ o , ␣ s , ␣ q,11 , ␣ z , and ␣ 12 , but not ␣ 13 were detected by this method (Fig. 3), showing that the anti-G protein antibodies coupled to the beads bind the ␣-subtypes of G proteins present in NK cell membranes. This binding was specific for anti-G protein antibodies, since no binding was observed with beads coated with normal rabbit serum (data not shown) or rabbit IgG (Fig. 3).
Reciprocally, when IL-2-activated NK cell membranes were immunoprecipitated with anti-NKR-P1, and then immunoblotted with either rabbit IgG as a control or with anti-G protein antibodies, the same G protein ␣-subunits were shown to associate with NKR-P1. Fig. 5 shows that 40-, 45-, 41-, and 40-kDa bands representing ␣ i3 , ␣ s , ␣ q,11 , and ␣ z , respectively, were associated with NKR-P1, while, ␣ i1,2 , ␣ o , ␣ 12 , or ␣ 13 were not. To demonstrate that anti-NKR-P1 utilized in the previous experiments described in Figs. 4 and 5 specifically binds NKR-P1 and not an unrelated molecule present in NK cell membranes, we have preformed more rigorous controls. In experiments described in Fig. 6A, NK cell membranes were immunoprecipitated with mouse anti-NKR-P1, rabbit IgG, mouse IgG, or mouse antibody to the CD8 molecule (OX8) present on the majority of rat NK cells. Upon immunoblotting with anti-NKR-P1, it was clear that this antibody bound to NKR-P1 present in the immunoprecipitate and not to OX8 or control mouse and rabbit IgG. Furthermore, NK cell membranes immunoprecipitated with anti-NKR-P1, but not those immunoprecipitated with mouse IgG, rabbit IgG, or OX8, were specifically immunoblotted with antibody to the common ␣subunit of G protein (Fig. 6B). These results clearly demonstrate that certain G protein ␣-subunits are coupled to NKR-P1 and not to other surface molecules such as OX8. DISCUSSION 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 (16), and were first characterized as activating receptors (15). Although the natural ligand for NKR-P1 is still undefined, 3.2.3 antibody induces redirected lysis (16), transduces signals important for regulating NK cell growth (17), and induces intracellular calcium mobilization (15), phosphoinositide turnover (15), and interferon-␥ secretion (17).
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 (25). This pathway is important for the activation of various secondary messengers such as phospholipase, in particular phospholipase C␤ (20,26), and the mitogen-activated protein kinase pathway (27). Recent work has shown that the ␤␥-dimer binds and activates the phosphatidylinositol 3-kinase ␥-isoform (27,28). In addition, this dimer binds pleckstrin homology domain (29), suggesting the importance of G proteins in mediating various biological activities inside the cells.
FIG. 6. Only NKR-P1, but not an unrelated surface molecule (OX8) is coupled to G proteins in NK cell membranes. A, membranes prepared from NK cells suspended in the solubilization buffer were immunoprecipitated overnight at 4°C with mouse antibody to OX8, NKR-P1, mouse IgG (MIgG), or rabbit IgG (RIgG). Protein A/Gagarose was added to the tubes, incubated for 4 h at 4°C, and then washed. The immunocomplexes were separated by 12% SDS-PAGE, electrotransferred, and then immunoblotted with anti-NKR-P1. B, similar to A except that the immunocomplexes were immunoblotted with antibody to the common ␣ of G protein instead of anti-NKR-P1. The results are representative of two different experiments.
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 (32); (ii) receptors present on human NK cells that recognize tumor targets, and are coupled to G s and G o (22), (iii) receptors present on rat NK cells that recognize tumor or allogeneic target cells, and are coupled to G o and G z (18), (iv) transforming growth factor-␤1 receptors present on rat NK cells, and are coupled to G o and G s (33), (v) the CXC chemokine IL-8 receptors present on human NK cells, and are coupled to G o (34), (vi) the CXC chemokine IP-10 receptors present on human IL-2-activated NK cells, and are coupled to G i , G o , and G q (35), (vii) the CXC chemokine SDF-1 receptors present on human NK cells, and are coupled to G o , G s , and G q (36), (viii) the CC chemokines MCP-1 and RANTES receptors present on human NK cells, and are coupled to G i , G o , G s , and G z (24), (ix) the C chemokine lymphotactin receptors present on human NK cells, and are coupled to G i , G o , and G q (35), and (x) exocytosis of NK cells, which involves certain G proteins (37).
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 ␣-subunits in NK cell membranes. Our results clearly demonstrate that G i3 , G s , G q , and G z , but not G i1,2 , G o , G 12 , or G 13 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 ␣subunit of G i3 , G s , G q , or G z 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 ␣-subunits of G i3 , G s , G q , or G z .
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-␤1 receptors (33,38), or insulin like growth factor-1 receptors (39) also bind G proteins. It is interesting that both transforming growth factor-␤1 type II receptors (40) and NKR-P1 receptors (10,11) 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.