INTRODUCTION

Two Ang II¹ receptor subtypes (AT₁ and AT₂) have been defined (1, 2). Most known physiological actions of Ang II are mediated by the AT₁ receptor (1, 2, 3, 4). On the other hand, the AT₂ receptor remains an enigma. Cloning of AT₂ receptor by our laboratory and others (5, 6, 7, 8) has revealed that like the AT₁ receptor, the AT₂ receptor also possesses a seven transmembrane domain structure, which is similar to that of the heterotrimeric guanine nucleotide binding protein (G protein)-coupled receptors (9). However, the biochemical association of this receptor to this class of protein has not been demonstrated.

Heterotrimeric G proteins, comprised of , , and  subunits, function as intermediates that couple cell surface receptors to intracellular effectors (10, 11). Much of the specificity of receptor action is dictated by the  subunit to which it is coupled. This subunit can be grouped into four major classes, each comprised of multiple members that are related by sequence homologies (s (s and olf), i (i1,i2 and i3, o, t1, t2, gust and z), q (q, 11, 14, 15, and 16) and the 12 (12 and 13)) (12). Interestingly, many seven-transmembrane receptors can, in fact, couple to several G proteins, increasing the complexity of the systems. Once thought to be only negative regulators of  subunit activity, the subunits have also been shown to be linked to the activation of various intracellular pathways (10, 11, 12).

In radioligand binding and in autoradiographic studies, the AT₁ receptor is known to be sensitive to GTP analogs (1, 2, 13), suggesting the coupling to G proteins. Stimulation of Ang II receptor in AT₁-rich tissues results in calcium mobilization through G_q/11 and modulation of cAMP levels through G_i (14, 15, 16). In contrast to the AT₁ receptor, in most tissues examined, binding to the AT₂ receptor is not susceptible to GTP analogs (1, 2, 13, 17, 18, 19), which has led to the suggestion that the AT₂ receptor does not interact with G proteins (13). However, indirect evidence has suggested that AT₂ receptor may also couple to G proteins (20, 21). In certain regions of the rat brain, binding to the AT₂ receptor was sensitive to GTP analogs and to pertussis toxin treatment (22). Similarly, in PC12w cells, the AT₂ receptor-mediated inhibition of phosphotyrosine phosphatase was reversed by pertussis toxin treatment (23). More recently, Kang et al. (24) reported that in vivo in cultured rat neurons, AT₂ receptor-mediated stimulation of delayed rectifier K⁺ current (I_k) could be abolished by intracellular application of an anti-G_i antibody.

The above data demonstrating sensitivity (or insensitivity) of radioligand binding to GTPS and pertussis toxin are indirect evidence for G protein coupling (or uncoupling). However, these approaches suffer from several caveats. Although the work of Kang et al. (24) is more direct, the use of cultured cells, which require several weeks of culture in order to develop an AT₂ receptor-mediated action, is also problematic. Therefore, the purpose of the present work was to demonstrate a direct, biochemical association of AT₂ receptor and its coupled G proteins in the rat fetus. The rat fetus was chosen because the AT₂ receptor is expressed at extraordinarily high levels in many fetal tissues.

EXPERIMENTAL PROCEDURES

Membrane fractions (100,000 × g pellet) were isolated as described (6, 7) from 18-day Sprague-Dawley rat whole fetus. To solubilize the receptor, the membrane pellets were resuspended in 25 mM sodium phosphate, pH 7.4, 5 mM EDTA, 5 mM EGTA, 200 mM KCl, 25% glycerol, 25 mM MgCl₂, plus 1% CHAPS (25). The 100,000 × g supernatant was collect and stored at 70 °C.

Membrane (10 µg of protein) or immunoselected fractions were incubated with 2 nM ¹²⁵I-Ang II (2,176 Ci/mM) at room temperature for 60 min in 100 µl of 20 mM Tris-HCl, pH 7.4, 10 mM MgCl₂, 0.25% bovine serum albumin. Bound and free ligands were separated by rapid filtration through GF/B Whatman glass fiber filters. For solubilized membranes, the filters were presoaked in 0.5% polyethylenimine. The filters were washed three times, and the bound radioligand was quantitated. Each point was performed in duplicate. Nonspecific binding was defined as radioactivity bound in the presence of 1 µM unlabeled ligand. For characterization of AT₂ receptor subtypes, the selective AT₁ receptor antagonist DuP 753 (losartan) and the selective AT₂ ligands CGP42112A or PD123319 (each at 1 µM) were employed.

Solubilized membrane samples in SDS-polyacrylamide gel (PAGE) buffer were incubated in boiling water bath for 5 min. Approximately 30 µg of protein was subjected to 10% SDS-PAGE and transferred onto nitrocellulose. Immunoblotting was carried out according to ECL Western blotting protocol (Amersham Life Science Inc.).

Solubilized AT₂ receptor preparations (1 ml of a 1 mg/ml solution) diluted in solubilization buffer containing 0.3% CHAPS were incubated with anti-G protein antibodies or antisera overnight at 4 °C with constant shaking (amounts of the antibody or antisera are indicated in the figure legends). Protein A-agarose (60 µl of 50% w/v) was then added and incubated with rocking for an additional 4 h at 4 °C. The samples were centrifuged at 14,000 rpm. The supernatant was removed, and the immunoselections were washed three times with 1 ml of ice-cold solubilization buffer containing 0.3% CHAPS. The AT₂ receptor in the immunoselection and in the supernatant was detected by radioligand binding assays as described above. In some experiments, 10 mM NaCl or 10 mM NaF plus 10 µM AlF₃ were added to the solubilized membrane preparations at the time of the immunoselection.

[³⁵S]GTPS Binding Assay

[³⁵S]GTPS binding was measured with a modification of assay described by Johnson and Corbin (26). Immunoselections were mixed with incubation buffer (20 mM Tris-HCl, 25 mM MgCl₂, 1 mM EDTA, 100 mM NaCl, and 1 mM dithiothreitol, pH 8) and 5 nM [³⁵S]GTPS (1, 332.0 Ci/mmol) in 100 µl. The binding was initiated by the addition of the immunoselections to the incubation buffer and was carried out for 1 h at 30 °C. The reactions were terminated by rapid filtration through GF/B Whatman glass fiber filters. The filters were washed three times with 5 ml of ice-cold buffer (20 mM Tris-HCl, pH 8.0, 25 mM MgCl₂, 100 mM NaCl) and then quantitated. Nonspecific binding was determined in the presence of 1 mM GTP and subtracted from total bound radioactivity.

Pertussis toxin was preactivated by incubation with 20 mM dithiothreitol, 0.125% SDS and 1 mg/ml BSA for 30 min at 30 °C. Solubilized membrane (30 µg) or immunoselections were incubated with pertussis toxin (10 µg/ml) and 5 µCi of [³²P]NAD in the presence or in the absence of 100 µM NAD for 1 h at 30 °C, in a final volume of 100 µl of reaction buffer (pH 7.4, 1 mM EDTA, 10 mM thymidine, 10 mM Hepes, 0.5 mM ATP, 0.1 mM GTP) (27, 28). The proteins were loaded into 10% SDS-PAGE gels. Gels were stained, dried, and autoradiographed at 70 °C for 5-24 h.

¹²⁵I-Ang II (2, 176 Ci/mmol) was purchased from Robert Speth at Washington State University. Dithiothreitol, GTPS, and CHAPS were from Sigma. Losartan was provided by Ronald Smith of DuPont-Merck. PD123319 was provided by Joan Keiser at Parke-Davis. CGP42112A was provided by Marc de Gasparo of Ciba-Geigy. ECL Western blotting detection reagents was from Amersham International Plc. Antisera sources are listed in Table I. (29, 30, 31, 32, 33).

Table I. Origins and specificity of antisera Antisera (code or catalog number) Specificity Source Reference I-355 Gi1 Susanne Mumby 29 J-883 Gi2 Susanne Mumby 29 3646 Gi1 David Manning 30 1521 Gi2 > Gz David Manning 30 371720 Gi1 Calbiochem 371729 Gi3 Calbiochem Gi1 Tomiko Asano 31 Gi2 Tomiko Asano 31 NEI-803 Gi3, Go Dupont NEN 32 06-270 Gi3, Go UBI 33 SC-386 (C-20) Gi1-3, Go, Gz, Gt Santa Cruz Biotech. SC-262 (C-10) Gi1, Gi2, Gi3 Santa Cruz Biotech. SC-387 (K-20) Go Santa Cruz Biotech. SC-388 (I-20) Gz Santa Cruz Biotech. SC-392 (C-19) Gq/11 Santa Cruz Biotech. SC-409 (S-20) G12 Santa Cruz Biotech. SC-410 (T-20) G13 Santa Cruz Biotech. SC-378 (T-20) G1, G2, G3, G4 Santa Cruz Biotech. IgG antirabbit IgG Sigma Nonimmune serum normal rabbit serum Amersham Int. PLC.

Data are presented as mean values ± S.E. Student's t test for unpaired observation and one-way analysis of variance were used for statistical evaluation. p < 0.05 was considered significant. Specific binding was calculated by subtracting nonspecific binding measured in the presence of unlabeled ligand from total binding. Saturation analysis were performed using nonlinear regression curve-fitting. Dissociation constant K_d and B_max were calculated from Scatchard plot using a linear regression program (Statview).

RESULTS

Binding of ¹²⁵I-Ang II to membranes prepared from 18-day-old rat fetus was of high affinity, saturable, and linearly proportional to the protein concentrations tested (1-60 µg, data not shown). Scatchard analysis revealed a single class of binding sites (Fig. 1A), yielding a K_d of 1.89 ± 0.11 nM and B_max of 1.91 ± 0.10 pmol/mg protein (n = 3). Nonspecific binding was determined in the presence of 1 µM CGP42112A and was less than 15% of total binding at the K_d of binding. Binding of ¹²⁵I-Ang II to the membrane preparation was competed by AT₂ selective ligand CGP42112A (91 ± 3.7% displacement at 1 µM, n = 3) and PD123319 (78 ± 3.1% displacement at 1 µM, n = 3) but was not sensitive to the AT₁-selective antagonist DuP 753 (7 ± 2.1% displacement at 1 µM, n = 3). (Fig. 1B).

We next tested the ability of detergents to solubilize the fetal membranes and release AT₂ receptors, which retain binding capacity. As shown in Fig. 1C, optimal solubilization of AT₂ receptor was achieved with 0.5-1% CHAPS, which yielded 70-60% of specific ¹²⁵I-Ang II binding and 50-60% of total protein. Because solubilization of AT₂ receptor with 1% CHAPS resulted in less nonspecific binding but higher AT₂ receptor binding in immunoselections (data not shown), 1% CHAPS was used in subsequent experiments.

¹²⁵I-Ang II binding to solubilized membrane preparations was specific and saturable, with moderate decrease in the affinity and B_max (Fig. 1A, K_d = 3.46 ± 0.12 nM, B_max = 1.33 ± 0.11 pmol/mg protein.). Competition studies with receptor specific ligands (losartan, PD123319, and CGP42112A) and nonsubtype selective ligands (Sar-1, Ile-8 Ang II, and Ang II) exhibited an inhibitory pattern similar to that seen in crude membrane preparation (Fig. 1B) and consistent with the pharmacology of the AT₂ receptor.

Presence of G Protein  and  Subunit Immunoreactivity in Solubilized Rat Fetus Membrane Preparation

Prior to an examination of the coupling of the AT₂ receptor to specific G proteins, we first assessed the expression of the various G proteins in the solubilized 18-day-old rat fetus membranes using Western analysis. A majority of the anti-G protein antibodies tested yielded strong signals with appropriate molecular sizes (Fig. 2). Specific immunoreactivity was detected at 40-41 kDa with G_i2 and G_i3 antibodies. Anti-G_q/11 antibody detected proteins of molecular masses of 42-43 kDa. Anti-G_z antibody recognized a major band at 41 kDa. Anti-G_common recognized a band at approximately 35-36 kDa. A weak band at 39 kDa was detected by anti-G_o antibody. However, several different anti-G_i1 antibodies as well as antibodies specific for G₁₂ and G₁₃ failed to detect protein, suggesting the low expression of these proteins in the whole rat fetus. Control immunoblots using IgG were negative (Fig. 2).

Immunoblotting of  and various  subunits of G proteins in solubilized rat fetus membrane preparation.

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Immunoselection of [³⁵S]GTPS Binding Proteins with Anti-G Protein Antibodies

In order to test the immunoselection procedure, the presence of GTP binding proteins in the immunoselections was examined by measuring the specific binding of [³⁵S]GTPS. Binding of [³⁵S]GTPS to both solubilized membrane preparation and to immunoselections was concentration-dependent and specific (data not shown). Under our experimental conditions, nonspecific binding defined in the presence of 1 mM of GTP was less than 10%. As shown in Fig. 3, polyclonal antibodies raised against several G proteins (G_i1-3, G_z, and G_q/11) were tested and shown to immunoselect, in a concentration-dependent manner, GTP binding proteins. Similar numbers of GTP binding sites were immunoselected with each antibody. The percentage of total GTP binding sites in the solubilized membrane that was immunoselected with the antibodies specific for G_i1-3, G_z, and G_q/11 were 1.8, 1.4, and 1.2%, respectively. This must be considered as an underestimate because solubilized membrane has been reported to contain GTP binding activities distinct from G proteins (34).

i1-3

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q/11

A second method was used to test the immunoselection procedure. Because G_i1-3 are known to be sensitive to pertussis toxin, the presence of pertussis toxin-sensitive G proteins was also investigated. Labeling of both solubilized membrane preparation and SC-262-immunoselected fractions but not IgG-immunoselected fractions with ³²P-NAD in the presence but not in the absence of pertussis toxin resulted in a radiolabeled band corresponding to molecular mass of approximately 40-41 kDa (Fig. 4). This band was not observed when 100 µM NAD was added, suggesting pertussis toxin-specific reaction of ADP-ribosylation (Fig. 4).

i1-3

We next tested the ability of antibodies raised against the G proteins to co-select angiotensin II binding sites. Initially, we used an anti-G_common antibody (SC-386), which interacts with G_i1-3, G_o, G_z, and G_t. The results demonstrate the selection of a low level of Ang II binding sites that were not observed with IgG (Fig. 5A). Next, antibodies more selective against specific G proteins were tested. Consistently, anti-G_i1-3 (SC-626) concentration-dependently co-immunoselect the solubilized Ang II binding sites (Fig. 5B). Interestingly, this antibody selected a higher amount of Ang II binding sites compared with G_common antibody. To address specificity, SC-262P, the peptide to which SC-262 was raised completely blocked the ability of this polyclonal antibody to immunoselect the Ang II binding sites. SC-262P itself did not interfere with ¹²⁵I-Ang II binding (Fig. 5C). No angiotensin II binding sites were co-immunoselected with control IgG. As shown in Fig. 5D, binding of ¹²⁵I-Ang II to SC-262-immunoselected samples was of high affinity and yielded saturation binding curves comparable with those observed with solubilized membrane preparations. Scatchard analysis revealed that the K_d of the immunoselected binding sites were comparable with that observed for the binding sites in the solubilized membrane fraction. In contrast, no specific Ang II binding sites were detected in immunoselections by anti-G, anti-G_z, anti-G_o, anti-G₁₃, anti-G₁₂, and anti-G_q/11 antibodies (Fig. 6), even though anti-G_q/11 and anti-G_z were almost as effective in concentration-dependently immunoselecting GTP binding proteins (Fig. 3).

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These Ang II binding sites represented AT₂ receptors. In experiments where specific binding was defined independently with Sar-1, Ile-8 Ang II, or CGP42112, the compounds were shown to compete to similar levels (Fig. 7). Moreover, DuP 753 was not able to compete ¹²⁵I-Ang II binding to the immunoselected material. In addition, binding with ¹²⁵I-CGP42112 was performed in selective experiments and demonstrated results comparable with those obtained with the ¹²⁵I-Ang II (data not shown).

i1-3

i1-3

The above results suggest the close association between the AT₂ receptor and G_i, which may be an actual coupling of the receptor to this G protein. Alternatively, this association may only indicate the presence of these proteins within the same mixed micelles, which are formed during the solubilization step. The latter explanation is unlikely because antibodies specific for several other G proteins failed to immunoselect AT₂ binding sites; the proteins in the mixed micelles should be randomly distributed. However, to provide further and more direct data concerning this point, we examined the ability of aluminum fluoride, which is known to disrupt G protein-receptor complexes, to inhibit the co-immunoselection of the AT₂ binding sites by the anti-G_i antibody. The addition of NaF plus AlF to the immunoselection decreased the ability of the anti-G_i antibody to immunoselect AT₂ binding sites by 38% (specific binding obtained with anti-G_i antibody, 1200 ± 66; anti-G_i antibody plus NaF plus AlF; 740 ± 98) However, NaCl at the same concentration was without effect. Thus, taken together, the results are consistent with a coupling of the AT₂ receptor and G_i.

To determine which subtype of G_i was involved, several anti-G_i1-, G_i2-, and G_i3-specific antibodies were tested. An anti-G_i2 antibody (J-883) co-immunoselected AT₂ receptor-G_i complexes but to an extent less than that selected with anti-G_i1-3 (SC-262) (Fig. 8). Similarly, an anti-G_i3 antibody (NEI-803) also co-immunoselected AT₂ receptor, again to an extent less than that observed with anti-G_i1-3 (Fig. 8). Several other anti-G_i subtype antibodies yield similar results (G_i2 (1521), 670 ± 93 cpm; G_i2 (Asano), 675 ± 83 cpm; G_i3 (06-270), 504 ± 67 cpm; G_i3 (371729), 431 ± 84 cpm). We next tested the ability of anti-G_i2 and anti-G_i3 antibodies to immunoselect Ang II binding sites in sequential experiments. Previous immunoreaction of the membrane preparation with anti-G_i3 (NEI-803) antibody did not affect the ability of anti-G_i2 (J-883) antibody to immunoselect Ang II binding sites. Similarly, previous immunoreaction of the membrane preparation with anti-G_i2 (J-883) antibody did not affect the ability of anti-G_i3 (NEI-803) antibody to immunoselect Ang II binding sites. On the other hand, prior treatment with anti-G_i1-3 (SC-262) antibody abolished the subsequent immunoselection with anti-G_i1-3 antibody (Table II).

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Table II. Association of the AT2 receptor with both Gi2 and Gi3 Round I experiments were performed by incubating solubilized rat fetus membrane with anti-Gi2 anti-Gi3 or anti-Gi1-3 or their appropriate controls (nonimmune serum, control IgG) overnight at 4 °C. Antibody-G protein complexes were then collected with 60 µl (50% w/v) protein A-agarose. The immunoselections were incubated with 125I-Ang II (2 nM) for 1 h at room temperature. Nonspecific binding was defined in the presence of 1 µM CGP42112A. The same procedure was followed with round II, except that the supernatants of round I were used. The data are presented as means ± S.E. (n = 2). NI, nonimmune serum. Round I Round II Antibody 125I-Ang II specific bound Antibody 125I-Ang II specific bound cpm cpm NI, 10 µl 139  ± 68 NI, 10 µl 120  ± 50 Anti-Gi2 (J-883), 10 µl 730  ± 97a Anti-Gi3 (NEI-803), 10 µl 690  ± 153a Anti-Gi3 (NEI-803), 10 µl 613  ± 81a Anti-Gi2 (J-883), 10 µl 701  ± 176a IgG, 10 µg 128  ± 90 IgG, 10 µg 189  ± 143 Anti-Gi1-3 (SC-262), 10 µg 1261  ± 172a Anti-Gi1-3 (SC-262), 10 µg 276  ± 86 a p < 0.05 with respect to the appropriate control.

Consistent with the very low levels of G_i1 observed in the 18-day-old rat fetal membranes by Western analysis, an anti-G_i1 antibody (I-355) was not capable of co-immunoselecting AT₂ binding sites (Fig. 8). Similar results were obtained with other anti-G_i1 antibodies (anti-G_i1 (3646), 277 ± 101 cpm; anti-G_i1 (371720), 303 ± 103 cpm; anti-G_i1 (Asano), 182 ± 28 cpm).

DISCUSSION

Based on the ability of highly specific antibodies raised against the G subunits to co-immunoselect AT₂ binding sites, we conclude that the AT₂ receptor is G protein-coupled, at least in the rat fetus. The specificity of this approach was provided by the fact that control IgG or nonimmune sera failed to select Ang II binding sites. Importantly, antibodies against G_q/11 and G_z also failed to immunoselect Ang II binding sites, despite their ability to immunoselect comparable numbers of GTPS binding sites. The immunoselected Ang II binding sites were of high affinity with saturation curve similar to that observed with solubilized membranes.

Ang II acts at multiple sites within the cardiovascular system to assist in the regulation of cardiovascular homeostasis (1, 2). These effects are mediated by high affinity Ang II binding sites, which, at least in the adult, have been shown to be of the AT₁ subtype (1, 2). This receptor is a seven transmembrane-spanning receptor (35) that is coupled primarily to G_q resulting in the activation of phospholipase C and the subsequent release of IP₃ and diacyl glycerol, leading to increased intracellular calcium and activation of protein kinase C (14, 15, 16). Alternatively, this receptor is coupled to G_i, resulting in the inhibition of adenylyl cyclase activity (14, 15). Activation of the AT₁ receptor induces a variety of actions depending on the tissue, exhibiting specific adrenal, renal, and vascular effects (1, 2, 3, 4).

In contrast to the AT₁ receptor, the AT₂ receptor remains a mystery. Initially described on the basis of selective binding to specific ligands, this receptor was found to be expressed at very low levels in the adult and only in selective tissues (1, 2). On the other hand, binding studies revealed that this receptor was highly expressed in embryonic, fetal, and neonatal tissues, which lead to the speculation that this receptor may mediate some undefined action of Ang II in the processes associated with growth, development, or differentiation (18, 36, 37). Ang II has been shown, both in culture as well as in vivo, to induce growth and alter cellular phenotype (39, 40). However, the ability of AT₁-specific antagonists to block these effects of Ang II, at least in the adult, argued against this role for the AT₂ receptor (41, 42). Recently, we have found that the AT₂ receptor mediates an antigrowth effect, counteracting the growth promoting effects mediated by the AT₁ receptor (43). These results were obtained in a variety of models and may depend on the AT₂-mediated decrease in MAP kinase activity.

The discovery that the AT₂ receptor shared the structural characteristics of the serpentine receptor family strongly suggested that this receptor may couple to G proteins (5, 6, 7, 8). Indirect evidence, such as the ability of pertussis toxin to block certain actions ascribed to the AT₂ receptor, was consistent with this suggestion (20, 21, 22, 23). In the course of our study, we became aware of the work of Kang et al. (24), who demonstrated that the AT₂ receptor mediated an activation of a potassium current in cultured neonatal rat neurons. Intracellular delivery of an anti-G_i antibody recognizing all of G_i1, G_i2 and G_i3 by micropipette abolished this current, suggesting a role for G_i. Although an elegant and well controlled approach, the intracellular delivery of antibodies suffers from certain caveats, the major being a lack of this approach to demonstrate direct receptor-G protein interaction. Moreover, the ability of Ang II to induce this potassium current is not found in freshly isolated neurons but only in cells cultured for more than 12 days. Thus the possibility that this observation represented what could happen and not necessarily what does happen in vivo remained. Our direct evidence of an AT₂ receptor-G_i1-3 interaction is therefore complimentary to their approach.

What are the intracellular signals induced by the AT₂ receptor by way of G_i (and associated )? It has been shown that all three G_i subtypes (G_i1, G_i2, and G_i3) are able to stimulate the G protein-gated atrial potassium channel and to inhibit adenylyl cyclase (44, 45, 46). The activation of the neuronal potassium current is consistent with the idea that G_i is involved as is the evidence that pertussis toxin will block certain actions of the receptor. Our evidence for an antiproliferative action mediated by the AT₂ receptor is harder to explain. Other results from our laboratory demonstrate that the activation of AT₂ receptor leads to a decrease in growth and an inhibition of mitogen-activated protein kinase activity (43). The results in the current study demonstrate the coupling (or the physical association) of the receptor to G_i; especially G_i2 presents a conundrum. An increasing body of evidence suggests that G_i2 stimulates growth via a stimulation of the mitogen-activated protein kinase pathway (47, 48, 49). It could be argued that the association of the AT₂ receptor and G_i2 may not occur in vivo but is an artifact of the extraction procedure exists; however, we have recently obtained immunohistochemical evidence that at least in the fetal aorta, the AT₂ receptor and G_i2 are expressed in the same area of the vessel wall.² Although compartmentalization of receptors and G proteins in different membrane microdomains exist in some cells (38), such compartmentalization has not been described in smooth muscle but is usually associated with epithelial cell types. Thus, we are left without a suitable explanation. One potential explanation is that in whole cells, the receptor is more strongly associated with the other  subtypes, G_i3 or perhaps even G_i1. Another is that the receptor-G_i2 interaction involves a unique subunit that is actually mediating the anti-growth response, overriding the stimulating effect of G_i2. Other explanations may involve the inhibition of G_i2 activity following stimulation of the AT₂ receptor or the association of the AT₂ receptor with a novel G_i, which cross reacts with the anti-G_i antibodies. Clearly, this area requires further study.

The direct evidence for the association of the AT₂ receptor and G_i brings up another issue, that of the lack of sensitivity to GTP analogs such as GTPS. It is known that G proteins cycle between an inactive (GDP bound form) and active (GTP bound) states (10, 11, 12). The GDP bound- subunit associates with subunits to form the heterotrimer, which is able to associate with ligand-free receptors. Receptors coupled to hetrotrimers usually bind ligand with high affinity. When the receptor is activated by agonist it undergoes a conformational change that is transmitted to the  subunit, promoting the exchange of GDP for GTP. This causes a dissociation of the  subunit both from the receptor and from and frees the  subunit to interact with its appropriate effector. Receptors not associated with hetrotrimers usually exhibit a lower affinity for agonist. Once GTP is cleaved to GDP, the  subunit ceases its effector stimulation, reassociates with , and subsequently can reassociate with the receptor. Substitution of GTP with a nonhydrolyzable analog such as GTPS or Gpp(NH)P forces the  subunit to remain in its active form. Subsequently, no reassociation with and receptor occurs, and the affinity of the receptor thus remains in a low affinity state. The only evidence for a GTPS shift in affinity state of the AT₂ receptor has been obtained in radioligand autoradiographic studies or certain brain nuclei (20, 21); all other published studies using whole cells or membrane fractions have been negative (1, 2, 13, 17, 18, 19). This inability of GTPS to shift the AT₂ receptor to a low affinity state has often been cited as evidence that the receptor in not G protein-coupled. The inability of GTPS to induce a low affinity shift of AT₂ receptors in membranes from whole rat fetus may suggest that a majority of the receptors are not G protein-coupled. Consistent with this, the co-immunoselection of the receptor using anti-G protein antibodies was inefficient, yielding only a small fraction of the total AT₂ binding sites. Alternatively, the AT₂ receptor may couple to G proteins by a nonconventional mechanism or may couple to a G protein with a low rate of GDP-GTP exchange. Consistent with this latter possibility, in our study, GTPS did not influence binding to the immunoselected AT₂ receptor nor did it inhibit the immunoselection of the receptor by the G protein antibody, whereas AlF, which binds to the GDP bound G protein, did attenuate the ability of G protein antibody to co-immunoselect AT₂ binding sites.

In conclusion, co-immunoselection AT₂ receptor with anti-G protein antibodies proved useful in identification of direct association of AT₂ receptor and its coupled G proteins. The present study provide the first biochemical evidence that AT₂ receptor is G protein-coupled. The demonstration that AT₂ receptor is able to couple to both G_i2 and G_i3 may enhance our understanding of the signal transduction pathways underlying the physiological functions mediated by AT₂ receptor stimulation.