INTRODUCTION

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 (1-3). 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 IP₃, the mobilization of intracellular calcium, the secretion of interferon-, and the degranulation and cytotoxicity of NK cells (15-17).

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

Breeding pairs from the rat strains of PVG were bred in our laboratory or were purchased from Harlan Olac Ltd. (Bichester, United Kingdom (UK)).

Leupeptin, aprotinin, pepstatin A, phenylmethylsulfonyl fluoride, dithiothreitol, Tris-HCl, HEPES, CHAPS, glycerol, KCl, sodium phosphate, EDTA, EGTA, MgCl₂, bovine serum albumin, and GTP were purchased from Sigma. RPMI 1640 medium, PBS, antibiotics, fetal calf serum, L-glutamine, nonessential amino acids, and 2-mercaptoethanol solution were from Life Technologies, Inc. (Paisley, Scotland).

Anti-NKR-P1 monoclonal antibody (3.2.3) was a generous gift from Dr. John C. Hiserodt (University of California, Irvine, CA). Anti-CD3 monoclonal antibody (G4.18) was a gift from Dr. Bruce M. Hall (Liverpool, Australia). Monoclonal mouse OX8 (reacting with rat CD8) was a gift from the Cellular Immunology Unit, Department of Pathology, Oxford University (Oxford, UK). Rabbit polyclonal anti-G protein antibodies AS/7 (anti-G_i1,G_i2), EC/2 (anti-G_i3), GC/2 (anti-G_o), RM/1 (anti-G_s), QL (anti-G_q,G₁₁), and GA/1 (anti-G_common ) were purchased from NEN Life Science Products (Brussels, Belgium). Anti-G_z was purchased from Gramsch Laboratories (Schwabhausen, Germany). Anti-G₁₂, anti-G₁₃, and goat anti-mouse HRP were from Santa Cruz Biotechnology (Santa Cruz, CA). Goat anti-rabbit HRP-conjugated antibody was from Bio-Rad. Normal rabbit serum (NRS) and rabbit IgG were from Sigma.

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 × g, 1.077 g/ml (Nycomed Pharma, Oslo, Norway). The cells were washed and were depleted of CD3⁺ 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 IgG₁ magnetic Dynabeads (Dynal, Oslo, Norway), precoated with mouse anti-rat NKR-P1 mAb (3.2.3), to positively select NKR-P1⁺ cells, which mark most NK cells. Positively selected NK cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 1% nonessential amino acids, 10 units/ml penicillin, 100 µg/ml streptomycin, and 5 × 10⁵ M 2-mercaptoethanol plus rat recombinant IL-2 equivalent to approximately 1000 IU/ml human IL-2, for 7-10 days. The cells contain more than 98% NKR-P1⁺, CD3, CD5, and TCR cells when examined by flow cytometry.

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 × 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 MgCl₂, 2 mM EDTA in addition to the enzyme inhibitors (40 µg/ml phenylmethylsulfonyl fluoride, 2 µg/ml pepstatin A, 10 µg/ml leupeptin, and 2 µg/ml aprotinin). After two steps of homogenization and sonication, the mixture was centrifuged at 1000 × g for 10 min at 4 °C. The supernatants were transferred to Beckman tubes and ultracetrifuged at 150,000 × g for 45 min at 4 °C. The pellets were suspended in a lysis buffer, snap-frozen, and stored at 80 °C.

GTP binding was measured using a method described by us (23, 24). About 50-100 µg of membrane proteins were incubated in 100 µl buffer containing 20 mM HEPES/NaOH, pH 7.4, 100 µM EDTA, 125 µM MgCl₂, and 10 nM [-³⁵S]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 µl of ice-cold buffer containing 100 mM Tris-HCl, pH 8.0, 25 mM MgCl₂, 100 mM NaCl, and 20 µM 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 µg/ml anti-NKR-P1 for 1.5 min at 37 °C were preincubated in a binding buffer containing 10 nM [-³⁵S]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 µg/ml anti-NKR-P1 for 1.5 min, added to the GTP binding buffer plus [-³⁵S] 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.

Immunoblotting was performed as described (18). Briefly, 100 µg 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).

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 MgCl₂, plus 1% CHAPS. They were centrifuged at 100,000 × g, and the supernatants were collected and stored at 80 °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.

Significant values were determined using a two-tailed Student's t test.

RESULTS

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 µg/ml anti-NKR-P1 (3.2.3 antibody) resulted in a maximum binding of [-³⁵S]GTP (p < 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 < 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⁺ 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).

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Selective Binding of Various G Protein -Subtypes to NKR-P1

To examine the nature of G protein -subtypes coupled to NKR-P1 molecules, 96-well plates were coated with goat anti-rabbit IgG and then incubated with anti-_i1,2, anti-_i3, anti-_o, anti-_s, anti-_q,11, anti-_z, anti-₁₂, and anti-₁₃, or NRS as a control. After activation of IL-2-activated NK cell membranes with anti-NKR-P1 (10 µg/ml for 1.5 min at 37 °C), they were transferred to these plates and incubated on ice. _i3, _s, _q,11, and _z but not _i1,2, _o, ₁₂, or ₁₃ in NK cell membranes significantly bound GTP after ligating NKR-P1 molecules (p < 0.01, 0.001, 0.001, and 0.002, respectively, as compared with the basal GTP binding). Control rabbit IgG or NRS did not induce any significant binding of GTP (Fig. 2A).

Determination of GTP binding to various G protein -subtypes by immunoselection methods.

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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 anti-rabbit coupled beads. Membranes from NK cells stimulated with anti-NKR-P1 (10 µg/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 < 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).

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Coimmunoprecipitation of NKR-P1 with G Protein -Subtypes

A coimmunoprecipitation assay was utilized to investigate whether there was any direct interaction between NKR-P1 and G proteins. IL-2-activated NK cell membranes were immunoprecipitated with various anti-G protein antibodies or with rabbit IgG (RIgG) as a control and then immunoblotted with anti-NKR-P1. The -subunits of G protein immunoprecipitated with anti-_i3, anti-_s, anti-_q,11, or anti-_z are associated with a 60-kDa band upon immunoblotting with anti-NKR-P1 (Fig. 4), while _i1,2, _o, ₁₂, or ₁₃ failed to associate with the 60-kDa band representing NKR-P1. Whereas mouse IgG failed to detect NKR-P1 when NK cell membranes were immunoprecipitated with mouse IgG (MIgG), mouse anti-NKR-P1 was able to detect NKR-P1 immunoprecipitated with anti-NKR-P1 (NKR-P1 in Fig. 4).

Physical coupling of NKR-P1 with various G protein -subtypes.

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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.

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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.

More than 20 -subunits and at least 5 - and 10 -subunits have been identified so far (30). The -subunit is divided into four subfamilies. These are (i) _s (stimulatory of adenylyl cyclases), which includes _s and _olf; (ii) _i (inhibitory of adenylyl cyclases), which includes _i1, _i2, _i3, _o, _t1, _t2, _z, and _gust; (iii) _q (activator of phospholipases), which includes _q, ₁₁, ₁₄, and ₁₅/₁₆; and (iv) ₁₂, which includes ₁₂ and ₁₃. In its resting state, the -subunit binds GDP and upon ligation of the receptors, conformational changes occur within the receptor -subunit initiating the activation of G proteins, resulting in the binding of GTP to the -subunit and its dissociation from the -dimer (30, 31). Both the -subunit and the -dimer can then interact with various regulatory effector molecules (30, 31).

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, 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 -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.