The alpha 2A-Adrenergic Receptor Discriminates between Gi Heterotrimers of Different beta gamma Subunit Composition in Sf9 Insect Cell Membranes*

Mark RichardsonDagger and Janet D. Robishaw§

From § Henry Hood Research Program, Pennsylvania State University College of Medicine, Weis Center for Research, Danville, Pennsylvania 17822-2614

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In view of the expanding roles of the beta gamma subunits of the G proteins in signaling, the possibility was raised that the rich diversity of beta gamma subunit combinations might contribute to the specificity of signaling at the level of the receptor. To test this possibility, Sf9 cell membranes expressing the recombinant alpha 2A-adrenergic receptor were used to assess the contribution of the beta gamma subunit composition. Reconstituted coupling between the receptor and heterotrimeric Gi protein was assayed by high affinity, guanine nucleotide-sensitive binding of the alpha 2-adrenergic agonist, [3H]UK-14,304. Supporting this hypothesis, the present study showed clear differences in the abilities of the various beta gamma dimers, including those containing the beta 3 subtype and the newly described gamma 4, gamma 10, and gamma 11 subtypes, to promote interaction of the same alpha i subunit with the alpha 2A-adrenergic receptor.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Consistent with the steadily increasing number of G protein1 beta  and gamma  subtypes that has been revealed in recent years (1), in vivo studies have indicated a role for this structural diversity in the specificity of signaling. In this regard, antisense studies by Kleuss et al. (2, 3) have demonstrated a specific requirement for the beta 1 and gamma 3 subunits in the somatostatin receptor signaling pathway in rat pituitary GH3 cells, with a similarly specific requirement for the beta 3 and gamma 4 subunits in the muscarinic receptor signaling pathway. Also, a ribozyme study by Wang et al. (4) has shown a specific involvement of the gamma 7 subunit in the beta -adrenergic receptor signaling pathway in human kidney 293 cells. Taken together, these in vivo studies indicate that the composition of the beta gamma dimer has important ramifications for the fidelity of signaling that is probably manifested at the level of the receptor.

A growing body of in vitro evidence supports a direct interaction between the receptor and the beta gamma dimer (5). In particular, direct interaction of transducin beta gamma with rhodopsin has been shown with a fluorescence energy transfer technique (6). This association was blocked by a synthetic peptide derived from the carboxyl-terminal tail of rhodopsin, suggesting a site of direct contact between beta gamma and rhodopsin. Moreover, cross-linking studies have confirmed a receptor contact site on the beta  subunit. A synthetic peptide derived from the carboxyl-terminal portion of the putative third cytoplasmic loop of the alpha 2A-AR could be cross-linked to the carboxyl-terminal region of the beta  subunit (7).

To date, in vitro studies examining the contribution of a limited number of beta gamma dimers to the specificity of receptor coupling have not yielded the same high degree of discrimination shown in the in vivo studies cited above (8, 9). The present study extended this analysis to the alpha 2A-adrenergic receptor and to beta gamma dimers that represent the most extensive degree of structural diversity examined to date. Since baculovirus expression has been shown to be an effective means for producing functional G protein subunits (10-13) as well as G protein-coupled receptors (8, 9, 14, 15), this system was used to measure the level of interaction between the recombinant alpha 2A-adrenergic receptor expressed in Sf9 cell membranes and reconstituted in the presence or absence of purified Gi proteins of varying beta  or gamma  subtype composition. Among the two beta  subtypes or eight gamma  subtypes tested, 30-fold differences were observed in their relative abilities to support coupling of the same alpha  subunit to the recombinant alpha 2A-adrenergic receptor. These data demonstrate that the specificity of alpha 2-adrenergic receptor-G protein interactions is affected by the beta gamma dimer composition.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression of alpha 2A-Adrenergic Receptor-- A pVL1392 transfer vector containing alpha 2A-adrenergic receptor cDNA was generously provided by Dr. H. Kurose and R. Lefkowitz (Duke University, Durham, NC). Recombinant baculovirus encoding the alpha 2A-adrenergic receptor was generated by co-transfection of alpha 2A-pVL1392 with a linearized lethal deletion mutant of Autographa californica as directed by the supplier (BaculoGold, PharMingen Corp.). Expression by recombinant baculovirus was identified by specific binding of the alpha 2-adrenergic radioligand, [3H]yohimbine (described below). A positive recombinant was isolated through four rounds of plaque purification. Receptors were expressed by inoculating Sf9 insect cells at an m.o.i. of 1 in IPL-41 medium, 1× lipid concentrate, and 1% heat-inactivated fetal bovine serum (Life Technologies, Inc.) at a density of 2 × 106 cells/ml. After 72 h, cell pellets were lysed by nitrogen cavitation (500 pounds/square inch for 30 min at 4 °C) in 100 ml of ice-cold lysis buffer (25 mM Tris, pH 7.4, 1 mM EDTA, 10 mM MgCl2, 100 mM NaCl, 0.02 mg/ml phenylmethylsulfonyl fluoride, 0.03 mg/ml leupeptin, and 1 mM benzamidine) and centrifuged at 4 °C for 10 min at 600 × g. The supernatant was centrifuged at 40,000 × g for 40 min at 4 °C. The pellets were resuspended, washed once in lysis buffer (40,000 × g, 40 min), and resuspended in 10 ml of lysis buffer. Protein concentration was determined by Coomassie assay (Pierce). Particulate fraction protein was snap-frozen with liquid N2 in aliquots of 300 µg each and stored at -80 °C. Receptor expression was quantitated by saturation binding of [3H]yohimbine, as described under "Experimental Procedures." A single 500-ml expression culture yielded adequate material to carry out all of the reconstitution experiments.

Expression and Purification of G Protein Subunits-- Recombinant baculoviruses directing the expression of beta 1, gamma 1, gamma 2, gamma 3, gamma 5, and gamma 7 recombinant baculovirus were described previously (16, 17). Isolation of human cDNAs encoding beta 3 (18) and gamma 4, gamma 10, and gamma 11 (19) was described previously. In these cases, recombinant baculoviruses were obtained by co-transfection of Sf9 insect cells with pVL1393 transfer vectors containing beta 3, gamma 4, gamma 10, or gamma 11 and a linearized lethal deletion mutant of A. californica nuclear polyhedrosis virus as directed by the supplier (BaculoGold, PharMingen). Recombinant viruses were identified by immunoblotting Sf9 cell lysates for expression of the appropriate subunits. Subtype-specific antibodies were generated as described previously (20) using the following synthetic peptides: gamma 4, CKEGMSNNSTTSIS (amino acids 2-14); gamma 10, CKDALLVGVPAGSNPFREPR (amino acids 45-63); and gamma 11, CPALHIEDLPEK (amino acids 2-12). Other subtype-specific antibodies have been described previously (20, 21). Recombinant virus encoding Galpha i1 containing a hexahistidine tag at amino acid position 121 was kindly provided by Dr. T. Kozasa (Southwestern Medical Center, Dallas, TX). One-liter cultures of Sf9 insect cells in IPL-41 medium, 1% heat-inactivated fetal bovine serum, and 1× lipid mix (Life Technologies, Inc.) were inoculated at a density of 2 × 106 cells/ml simultaneously with recombinant baculoviruses encoding alpha , beta , and gamma  subunits as follows: 6hisalpha i1 at m.o.i. = 2, beta 1 or beta 3 at m.o.i. = 3, and each of the gamma  subtypes at m.o.i. = 3. Under this condition, those gamma  subtypes predicted to contain a C-20 geranylgeranyl group are appropriately modified. However, those few gamma  subtypes predicted to contain a C-15 farnesyl group are variably modified at high levels of protein expression (10). Therefore, to optimize the addition of a C-15 farnesyl moiety, cultures of Sf9 cells expressing the gamma 1 and gamma 11 subtypes were also infected with recombinant baculovirus encoding both subunits of the mammalian farnesyltransferase at m.o.i. = 0.2. This virus was kindly provided by Dr. Thomas Kost (Glaxo Corp.). Cultures of Sf9 cells infected with the farnesyltransferase virus displayed greater than 15-fold higher activity toward the Ha-Ras fusion protein substrate than cultures not so infected. Moreover, cultures of Sf9 cells infected with the farnesyltransferase virus resulted in the majority of the gamma 1 and gamma 11 subtypes being modified with the C-15 farnesyl moiety, as shown previously (10). Expression of G protein beta  and gamma  subunits in particulate fractions was confirmed 72 h later by immunoblotting with subtype-specific antibodies (22).

Recombinant beta gamma heterodimers were purified to apparent homogeneity using the procedure described by Kozasa and Gilman (11) for purification of beta 1gamma 2. Following cholate extraction of particulate fractions, the cholate-soluble protein was diluted to 0.2% sodium cholate with 20 mM Hepes, pH 8.0, 100 mM NaCl, 1 mM MgCl2, 10 mM beta -mercaptoethanol, 10 µM GDP, and 0.5% polyoxyethylene 10-lauryl ether. The cholate-soluble extract was loaded onto a 4-ml Ni-NTA resin bed at 3-4-bed volumes/h (4 °C) and washed with 100 ml of the same buffer containing 300 mM NaCl and 5 mM imidazole. beta gamma dimers were eluted from the column by activation of 6hisalpha beta gamma with AMF (30 µM AlCl3, 50 mM MgCl2 and 10 mM NaF)-containing buffer: 20 mM Hepes, pH 8.0, 50 mM NaCl, 10 mM beta -mercaptoethanol, 10 µM GDP, 1% sodium cholate, 5 mM imidazole, 50 mM MgCl2, 10 mM NaF, and 30 µM AlCl3. The peak beta gamma -containing fractions were identified by immunoblotting for both beta  and gamma  subunits and then pooled and diluted to less than 10 mM NaCl using 20 mM Hepes, pH 8.0, 1 mM EDTA, 3 mM DTT, 3 mM MgCl2, and 0.7% CHAPS. beta gamma subunits were further purified by fast protein liquid chromatography on a Mono Q column (Amersham Pharmacia Biotech, HR 5/5) eluted with a linear NaCl gradient from 0 to 400 mM. Peak fractions were again confirmed by immunoblotting. The elution peaks were pooled and dialyzed overnight (3 buffer changes) against 20 mM Hepes, pH 8.0, 1 mM EDTA, 3 mM DTT, 3 mM MgCl2, 100 mM NaCl, and 0.7% CHAPS (Spectra/Por tubing, 6000-8000 molecular weight cut-off, Spectrum Medical Industries, Houston, TX). A mixture of beta gamma subunits purified from bovine brain by a previously described method (23) was also further purified on a Mono Q column by the same procedure. Following dialysis, purified beta gamma subunits were concentrated to approximately 0.1 mg/ml in an Amicon ultrafiltration device (PM10 membrane), and the final protein concentrations were determined by staining with Amido Black. Purified beta gamma subunits were snap-frozen in small aliquots with liquid N2 and stored at -80 °C.

The 6hisalpha i1 subunit was expressed alone (m.o.i. = 3) in a 1-liter culture of Sf9 insect cells for subsequent purification. Protein extraction, loading, and washing of the Ni-NTA column were identical to that described for beta gamma subunits. The 6hisalpha i1 was eluted from the Ni-NTA column with the following buffer, 20 mM HEPES, pH 8.0, 100 mM NaCl, 10 mM beta -mercaptoethanol, 1 mM MgCl2, 0.5% polyoxyethylene 10-lauryl ether, 10 µM GDP, and 150 mM imidazole, and was subsequently purified further on a Mono Q column (fast protein liquid chromatography) using the same procedure described for beta gamma subunits with the exception that collection tubes contained an aliquot of GDP to yield a final concentration of 10 µM GDP in each of the column fractions. Subsequent handling of 6hisalpha i1 was identical to beta gamma subunits.

Reconstitution of Receptor-G Protein Coupling-- Purified alpha  and beta gamma subunits were combined in 20 mM Hepes, pH 8.0, 1 mM EDTA, 3 mM DTT, 10 µM GDP, 0.02% sodium cholate to allow formation of G protein heterotrimers of defined subtype composition. The mixture was incubated in a total volume of 30 µl on ice prior to reconstituting the G proteins into Sf9 cell plasma membranes. An aliquot of the Sf9 membrane preparation expressing alpha 2A-adrenergic receptor was thawed and diluted to approximately 0.5 mg of protein/ml in 25 mM Tris, pH 7.6, 1 mM EDTA, 10 mM MgCl2, 1 mM benzamidine, 1 µg/ml pepstatin A, and 1 µg/ml aprotinin; then Gi heterotrimers were added to the membranes at the desired ratio of G protein to receptor and incubated on ice for 30 min prior to receptor binding assays. The CHAPS concentration during this incubation was <= 0.04%.

ADP-ribosylation Assay-- To assess the relative affinities of the various beta gamma dimers for the alpha  subunit, 330 ng of the various purified beta 1gamma dimers were combined with 500 ng of purified 6hisalpha i1 at a final ratio of 50:75 (mol:mol) of beta gamma :alpha . At this ratio, differences in the relative affinities of certain beta gamma dimers for the receptor were detected. As described previously (24), incubation of the resulting Gi heterotrimers was carried out in the presence of 200 ng of islet activating protein and [32P]NAD at 30 °C for 20 min and then terminated by precipitation with 30% trichloroacetic acid followed by rapid filtration over BA85 nitrocellulose filters. ADP-ribosylation of 6hisalpha i1 was measured by scintillation counting to detect [32P] bound to filters.

Radioligand Binding Assays-- [3H]UK-14,304 and [3H]yohimbine binding incubations were carried out in a total volume of 250 µl at 24 °C for 60 min in a reaction buffer consisting of 25 mM Tris, pH 7.6, 1 mM EDTA, 10 mM MgCl2 (plus 100 mM NaCl in assays of [3H]yohimbine binding). Radioligand binding was initiated by addition of 2-10 µg of Sf9 membrane protein and terminated by addition of 3 ml of the same ice-cold reaction buffer followed by rapid filtration over Whatman GF/C glass fiber filters on a Millipore filtration apparatus. Filters were rinsed twice more with the same buffer. Bound radioligand was quantitated by liquid scintillation counting (Beckman LS6500). Nonspecific binding was determined in the presence of 100 µM yohimbine. Statistical analysis of coupling (high affinity [3H]UK-14,304 binding) supported by different gamma  subtypes was done by a two-tailed, Student's t test for comparison of the variation between two means, rho  <=  0.05 indicated statistical significance.

Materials-- [imidazoylyl-4,5-3H]UK-14,304, [methyl-3H]yohimbine, [32P]nicotinamide adenine dinucleotide, and G protein beta -common antiserum (SW/1) were obtained from NEN Life Science Products; Ni-NTA-agarose was obtained from Qiagen (Chatsworth, CA); oxymetazoline was obtained from Research Biochemicals International (Natick, MA); ECL reagent and horseradish-linked anti-rabbit IgG were obtained from Amersham Pharmacia Biotech (Buckinghamshire, UK); and other chemicals were obtained from Sigma.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Characterization of alpha 2A-Adrenergic Receptor Expression in Sf9 Insect Cell Plasma Membranes-- Expression of the alpha 2A-adrenergic receptor in Sf9 insect cells was initially assessed using the antagonist [3H]yohimbine. Uninfected cells did not express the alpha 2A-adrenergic receptor (open circles, Fig. 1A). However, a significant level of the alpha 2A-adrenergic receptor was detected by specific [3H]yohimbine binding at 48 h after infection of cells with the alpha 2A-AR baculovirus (filled squares, Fig. 1A). The KD values for the recombinant receptor calculated by Scatchard analysis of the saturation binding curve ranged from 3.5 to 6 nM with Bmax values ranging from 2.7 to 13.3 pmol/mg protein depending on the cell preparation (inset of Fig. 1A). This KD range of the recombinant receptor was consistent with that of the native alpha 2A-adrenergic receptor (for review, see Ref. 25). Moreover, this moderate Bmax value was optimal for the purpose of this study since this level of recombinant receptor expression was readily measurable but required minimal amounts of purified G proteins for reconstitution. Finally, the properties of the recombinant alpha 2A-adrenergic receptor were characteristic of those of the native receptor. The specific [3H]yohimbine binding showed a NaCl sensitivity that is typical of the native receptor (25), i.e. the Bmax binding plateau was 45-55% lower in the presence of 100 mM NaCl (data not shown). Prazosin and oxymetazoline displaced the specific [3H]yohimbine binding from the recombinant receptor with the same order of potency of the native receptor (KD values of 4 and 0.022 µM, respectively). Taken together, these data confirmed the suitability of Sf9 cells for the expression of recombinant alpha 2A-adrenergic receptor that is functionally similar to the native receptor (26).


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Fig. 1.   Expression of alpha 2A-AR in Sf9 insect cell membranes. Sf9 insect cell plasma membranes were prepared following receptor expression, and [3H]yohimbine binding was assayed as described under "Experimental Procedures." Triplicate receptor binding incubations contained 5 µg of membrane protein each; data points are mean values ± S.E. A, black-square, specific [3H]yohimbine binding (total minus binding in the presence of 100 µM unlabeled yohimbine) in membranes following alpha 2A-AR expression; open circle , specific [3H]yohimbine binding in control membranes (no recombinant baculovirus); B, binding of 1.5 nM [3H]yohimbine in the presence of the indicated concentrations of competing ligand; triplicate incubations containing 10 µg of Sf9 plasma membrane and 10 µM GTPgamma S were handled as described under "Experimental Procedures." Curves were fit to the data by non-weighted, non-linear regression analysis using a one-site competition formula. Symbols are defined as follows: , displacement of [3H]yohimbine from alpha 2A-AR by prazosin (KD = 4 µM); black-square, displacement by oxymetazoline (KD = 0.022 µM). KD values were derived from the EC50 using the relationship described by Cheng and Prusoff (38).

Reconstitution of alpha 2A-Adrenergic Receptor-G Protein Coupling in Sf9 Cell Plasma Membranes-- Coupling of the alpha 2A-adrenergic receptor was examined in the presence and absence of exogenous Gi protein using the agonist [3H]UK-14304. The direct agonist binding technique is generally considered to be more sensitive than agonist displacement studies for detecting receptor-G protein complexes (25). When Sf9 cell membranes expressing the recombinant alpha 2A-adrenergic receptor were incubated in the absence of exogenous Gi protein, a low level of specific [3H]UK-14304 binding was detected, accounting for ~15% of the binding that was later observed in the presence of added Gi protein (Fig. 2). By contrast, when Sf9 cell membranes expressing the recombinant alpha 2A-adrenergic receptor were incubated in the presence of exogenous Gi protein (at a molar ratio of 100:1 Gi:receptor), the level of specific [3H]UK-14304 binding was increased by more than 5-fold, representing coupling of the recombinant receptor to the added Gi protein (Fig. 2). Moreover, the increased level of [3H]UK-14304 binding was reversed by the addition of GTPgamma S, reflecting uncoupling of the recombinant receptor from the added Gi protein. Thus, reconstituted coupling was easily distinguishable from the background coupling in this experimental system, thereby confirming the suitability of this experimental system for measuring the coupling of the recombinant receptor to added Gi proteins of varying beta gamma composition. For optimal resolution between the reconstituted and background coupling, a 4 nM concentration of [3H]UK 14,304 was used in subsequent experiments.


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Fig. 2.   Guanine nucleotide-sensitive [3H]UK-14,304 binding by alpha 2A-AR expressed in Sf9 insect cell membranes. Triplicate binding incubations containing 2.2 µg of membrane protein each were done in the absence or presence of 0.1 mM GTPgamma S at the radioligand concentrations indicated. , specific binding of [3H]UK-14,304 following reconstitution with purified Gi heterotrimer at a ratio of 50:1, Gi to receptor; open circle , binding in the presence of 100 µM GTPgamma S following reconstitution with Gi; black-square, specific binding without added Gi; , binding in the presence of GTPgamma S with no added Gi. A curve was fit to the data by non-weighted, nonlinear regression analysis using a one-site hyperbola fit. Gi heterotrimer (9.25 pmol) was composed of purified 6hisalpha i1 and purified bovine brain beta gamma (3:2, mol alpha /mol beta gamma ). Nonspecific binding (presence of 100 µM yohimbine) was virtually identical to low affinity binding (presence of 100 µM GTPgamma S); thus essentially all the radioligand binding within this concentration range represents high affinity, receptor-G protein complexes.

Receptor to G Protein Stoichiometry-- The requirements of the [3H]UK-14,304 binding assay for the G protein alpha  and beta gamma subunits were examined further. Consistent with previous studies of the A1 adenosine receptor (9), the combined interaction of both the G protein alpha  and beta gamma subunits was required in order to detect the high affinity state of the recombinant alpha 2A-adrenergic receptor with this binding assay. As shown in Fig. 3A, when the recombinant receptor was reconstituted with the 6hisalpha i1 subunit alone (at a molar ratio of 50:1 alpha :receptor), the level of [3H]UK-14304 binding was low and was indistinguishable from that observed with no added Gi heterotrimer. This result attests to the validity of the [3H]UK-14,304 binding assay to evaluate differences in the ability of beta gamma dimers of varying composition to induce the high affinity state of the recombinant receptor. Next, the recombinant receptor was reconstituted with a constant amount of 6hisalpha i1 subunit and increasing amounts of beta gamma dimer. As shown in Fig. 3B, raising the amount of beta gamma dimer increased the fraction of receptor in the high affinity state as measured by the higher level of [3H]UK-14,304 binding. When the amounts of beta gamma dimer and 6hisalpha i1 subunit approached a 1:1 ratio, the level of [3H]UK-14,304 binding reached a plateau, accounting for ~60% of the total receptor population as determined by [3H]yohimbine binding. A similar, maximal level of coupling was observed previously for the A1 adenosine receptor (9), suggesting that not all of the recombinant receptors are accessible for reconstitution with added G proteins. Under these conditions, any observed differences in the magnitude of [3H]UK-14,304 binding can be assumed to be attributable to selective interactions of beta gamma dimers of varying composition with the receptor rather than to alterations in G protein alpha -beta gamma subunit interactions.


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Fig. 3.   Optimization of receptor to G protein stoichiometry for reconstitution of high affinity, guanine nucleotide-sensitive [3H]UK-14,304 binding. A, binding of 4 nM [3H]UK-14,304 by alpha 2A-AR in Sf9 insect cell membranes assayed following reconstitution with heterotrimeric Gi, Galpha i subunit alone, or a mock reconstitution with no added G protein. Triplicate receptor binding incubations contained 2 µg each of membrane protein in the absence (filled bars) or presence (cross-hatched bars) of 0.1 mM GTPgamma S, yielding mean ± S.E. values. B, black-square, [3H]UK-14,304 binding by alpha 2A-AR reconstituted with alpha /beta gamma mixtures containing a constant amount of purified 6hisalpha i1 incubated (2 h on ice) with increasing amounts of purified bovine brain beta gamma to yield a final 6hisalpha i1:receptor ratio of 75:1 (mol/mol) throughout the curve and the beta gamma :receptor ratio indicated on the ordinate. The curve was fit to the data by non-weighted, non-linear regression analysis using a one-site hyperbola (GraphPad Prism). The data are representative of three similar results.

Purification of G Protein beta 1gamma Dimers-- To produce recombinant beta gamma dimers of varying gamma  composition, the beta 1 subtype was chosen since it has previously been shown to interact with all of the gamma  subtypes (17, 27, 28). The recombinant beta gamma dimers were purified using a procedure originally described by Kozasa and Gilman (11). Sf9 cells expressing the 6hisalpha i1, beta 1, and one of the following gamma 1, gamma 2, gamma 3, gamma 4, gamma 5, gamma 7, gamma 10, or gamma 11 subunits were prepared. Then, cholate-solubilized membrane extracts from these cells were bound to Ni-NTA agarose columns by virtue of the 6-histidine tag on the alpha i1 subunit; the beta gamma dimers were eluted from the columns by activating the bound heterotrimers with AMF; and the 6hisalpha i1 subunit was subsequently eluted from the columns with high imidazole. Further purification of the recombinant beta gamma dimers and the 6hisalpha i1 subunit was achieved by applying their enriched fractions from the Ni-NTA columns to Mono Q columns.

The purity of the 6hisalpha i1 subunit was assessed by SDS-PAGE and silver staining (29). As shown in Fig. 4A, the purified 6hisalpha i1 preparation contained one major band of the size expected for the alpha i1 subunit taking into account the added amino-terminal tag. The purity of the recombinant beta gamma dimers was also compared by SDS-PAGE and silver staining (Coomassie was used in the case of beta 1gamma 1). As shown in Fig. 4A, each purified beta gamma preparation was composed of two predominant bands by protein staining as follows: a 36-kDa band representing the beta 1 subunit, and a 5-8-kDa band representing one of the following gamma 1, gamma 2, gamma 3, gamma 4, gamma 5, gamma 7, gamma 10, or gamma 11 subunits. The identity of each beta gamma purified preparation was confirmed by immunoblotting with antibodies specific for each gamma  subtype. Antibodies specific for the gamma 1, gamma 2, gamma 3, gamma 5, and gamma 7 subunits were used for this purpose previously (20). However, antibodies specific for the newly described gamma 4, gamma 10, and gamma 11 subunits needed to be generated against synthetic peptides based on the unique amino acid sequences of these proteins. As shown in Fig. 4B, the identities of the purified beta 1gamma 4, beta 1gamma 10, and beta 1gamma 11 preparations were confirmed by immunoblotting with these antibodies.


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Fig. 4.   Purified recombinant G protein subunits. A, 1st lane, 3 µg of purified beta 1gamma 1 was trichloroacetic acid-precipitated and loaded onto a 15% polyacrylamide gel in 40 µl of Laemmli sample buffer and stained with Coomassie Blue following electrophoresis; 2nd to 9th lanes, silver-stained purified G protein subunits. 300 ng each (determined by Amido Black staining) of purified G protein subunits were trichloroacetic acid-precipitated and loaded onto 15% polyacrylamide gels in 40 µl each of Laemmli sample buffer containing 120 mM DTT. Gels were silver-stained following the method of Fawzi et al. (30). Lanes contain the following subunits: 2nd lane, beta 1gamma 2; 3rd lane, beta 1gamma 3; 4th lane, beta 1gamma 4; 5th lane, beta 1gamma 5; 6th lane, beta 1gamma 7; 7th lane, beta 1gamma 10; 8th lane, beta 1gamma 11; 9th lane, 6hisalpha i1. B, following Western transfer, 300 ng each of purified beta gamma subunits were probed with the following gamma -specific antibodies: gamma 4, E59 at 1/250; gamma 10, E57 at 1/250; and gamma 11, E60 at 1/300. The peak of beta gamma elution occurred at Mono Q fractions 11 and 12, corresponding to 200-240 mM NaCl, in each case.

Comparison of beta gamma Dimers of Varying gamma  Composition in Terms of Coupling to the alpha 2A-Adrenergic Receptor-- Gi heterotrimers of varying gamma  composition were tested for their relative abilities to couple with receptor as measured by the level of high affinity [3H]UK-14,304 binding. As shown in Fig. 5, all combinations of the 6hisalpha i1 subunit with the various beta gamma dimers were capable of inducing high affinity [3H]UK-14,304 binding, with the level of binding showing dependence on the gamma  composition. In all cases, the [3H]UK-14,304 binding was completely abolished by addition of GTPgamma S (data not shown). The beta 1gamma 1 dimer supported only a very low level of coupling to the recombinant alpha 2A-adrenergic receptor. By contrast, the beta 1gamma 11 dimer produced a high level of coupling to the recombinant alpha 2A-adrenergic receptor. Since the gamma 1 and gamma 11 subtypes are closely related, showing 76% identity (19), the observation that they promote very different levels of coupling suggests that the relatively small number of amino acid differences between the two subtypes are important for recognition by the receptor. The beta 1gamma 2, beta 1gamma 3, beta 1gamma 4, and beta 1gamma 7 dimers also produced high levels of coupling with the recombinant alpha 2A-adrenergic receptor. These four gamma  subtypes are closely related, showing 66-74% homology at the amino acid level, and therefore, the finding that they produce essentially identical levels of coupling is not unexpected. Finally, the beta 1gamma 5 and beta 1gamma 10 dimers yielded intermediate levels of coupling with the recombinant alpha 2A-adrenergic receptor. These two subtypes are only distantly related, showing less than 53% homology to each other or to other gamma  subtypes. Taken together, these results showed measurable differences between beta gamma dimers of varying gamma  composition to support coupling of the same alpha  subunit to the recombinant alpha 2A-adrenergic receptor. Statistical analysis revealed the beta 1gamma 1, beta 1gamma 5, and beta 1gamma 10 dimers supported the lowest levels of coupling. Previously, several groups have reported that the beta 1gamma 1 dimer was less effective than other beta gamma dimers in supporting coupling to numerous receptors (8, 9, 30) and that this problem could not be overcome by increasing its concentration. To extend further these observations, we showed that increasing the concentration of the beta 1gamma 5 dimer did not raise the level of receptor coupling (Fig. 5B), indicating the gamma 5 subtype has a lower intrinsic ability to interact with the recombinant alpha 2A-adrenergic receptor. Based on results for both the beta 1gamma 1 and beta 1gamma 5 dimers, a similar result could be predicted for the moderately effective beta 1gamma 10 dimer.


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Fig. 5.   Comparison of reconstituted alpha 2A-AR coupling with Gi heterotrimers containing different beta gamma subtypes. A, the 6hisalpha i1 subunit was incubated with each purified beta gamma dimer for 2 h on ice. The resulting Gi heterotrimers were reconstituted into an Sf9 cell membrane preparation expressing the recombinant alpha 2A-AR at a Bmax of 2.7 pmol/mg protein. This condition represents a ratio of alpha :beta gamma :receptor of 75:50:1. Receptor-G protein coupling was assayed by binding of 3.5 nM [3H]UK-14,304 ± 0.1 mM GTPgamma S in triplicate to obtain mean ± S.E. values. The data show total binding minus binding in the presence of GTPgamma S where background coupling (obtained without added G protein) has been subtracted from each value. The data shown are an average of three such experiments. Asterisks indicate a significant difference (rho  <=  0.05, by two-tailed Student's t) in agonist binding relative to purified bovine brain beta gamma . B, the 6hisalpha i1 subunit was incubated with increasing concentrations of the beta 1gamma 5 dimer for 2 h on ice and then reconstituted into an Sf9 cell membrane preparation expressing the recombinant alpha 2A-AR at a Bmax of 13.3 pmol/mg protein. The ratio of alpha :receptor is 75:1, and the ratio of beta 1gamma 5:receptor is shown on the ordinate. Receptor-G protein coupling was assayed by binding of 6 nM [3H]UK-14,304 ± 0.1 mM GTPgamma S in triplicate to obtain mean ± S.E. values. The data show total binding minus binding in the presence of GTPgamma S where background coupling (obtained without added G protein) has been subtracted from each value.

The lower activities of the beta 1gamma 1, beta 1gamma 5, and beta 1gamma 10 dimers could be due to differences in affinities for the alpha  subunit of the G protein rather than for the alpha 2A-adrenergic receptor itself. To evaluate this possibility, the affinities of representative beta gamma dimers for the 6hisalpha i1 subunit were measured by the pertussis toxin-dependent ADP-ribosylation assay. Under the reconstitution conditions used in this study, which employed relatively high concentrations of beta gamma dimers, there were no real differences in the affinity of these beta gamma dimers for the 6hisalpha i1 subunit (Fig. 6). Thus, the observed differences between beta gamma dimers of varying gamma  composition reflect their intrinsic abilities to interact with the receptor, suggesting structural diversity among gamma  subtypes plays a role in agonist-stimulated receptor-G protein complex formation.


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Fig. 6.   Islet activating protein-catalyzed ADP-ribosylation of 6hisalpha i1 in the presence of different beta gamma subtypes. Using the same conditions employed for the reconstitution in Fig. 5, 500 ng of purified 6hisalpha i1 were combined with 330 ng of the various purified beta 1gamma dimers at a final ratio of 75:50 (mol:mol) of alpha :beta gamma . Incubation of the purified proteins in the presence of 200 ng of islet activating protein and [32P]NAD was terminated after 20 min at 30 °C by precipitation with 30% trichloroacetic acid followed by rapid filtration over BA85 nitrocellulose filters. ADP-ribosylation of 6hisalpha i1 was measured by scintillation counting to detect [32P] bound to filters. Bars show the mean cpm ± S.E. values from triplicate determinations with the specific beta 1gamma combination indicated. Background [32P] bound to filters in the absence of 6hisalpha i1 has been subtracted from these values.

Purification of G Protein beta 3gamma Dimers-- A variety of approaches has been used to examine the ability of the 6 beta  and 11 gamma  subtypes to form beta gamma dimers (17, 19, 27, 28). Overall, these approaches have provided consistent results showing that all the known gamma  subtypes are able to interact with the beta 1 subtype and, to a lesser extent, the beta 2 subtype. However, these approaches have yielded conflicting results regarding the abilities of the known gamma  subtypes to interact with the beta 3 subtype. In this regard, the lack of a beta gamma dimer containing the beta 3 subtype to serve as a positive control in the various assays has been a particular hindrance. To this end, the method of Kozasa and Gilman (11) was used to obtain a beta gamma dimer containing the beta 3 subtype. Sf9 cells were infected with recombinant viruses encoding the 6hisalpha i1, beta 3, and gamma 5 subunits either simultaneously or separately, and the beta 3gamma 5 dimer was then purified by the procedure described above. As shown in Fig. 7A, the co-expression of the 6hisalpha i1, beta 3, and gamma 5 subunits resulted in the appearance of beta 3 and gamma 5 subunits in the AMF activation fractions as detected by immunoblotting. This result was consistent with the release of beta 3gamma 5 dimer from the 6hisalpha i1 subunit in response to AMF activation. As shown in Fig. 7B, the identity of the AMF-released beta  subunit as the beta 3 subunit was confirmed by immunoblotting with a beta 3 subtype-specific antibody (B-34). Taken together, these results supported the conclusion that the beta 3 and gamma 5 subunits interact to form a functional beta gamma dimer.


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Fig. 7.   Elution of beta 3gamma 5 from Ni-NTA agarose column. 100-ml cultures of Sf9 insect cells were inoculated with recombinant baculovirus encoding 6hisalpha i1, beta 3, and/or gamma 5. Cholate-soluble particulate protein was loaded onto a Ni-NTA agarose column that was washed and eluted as described under "Experimental Procedures." Western blot was performed on aliquots of the column fractions. A, simultaneous expression of 6hisalpha i1, beta 3, and gamma 5. The upper portion with beta -common antibody (SW/1, 1:5000) the lower portion with gamma 5-specific antibody (E56, 1:250). 1st lane, 30 µg of cholate-soluble particulate fraction; 2nd lane, 30 µg, column flow-through; 3rd lane, 30 µg, 5 mM imidazole wash at 4 °C; 4th lane, 30 µg, 5 mM imidazole wash at room temperature (R.T.); 5th to 9th lanes, 3 µg, elution by AMF; 10th and 11th lanes, 3 µg, elution by 150 mM imidazole. B, recognition of purified beta 3gamma 5 dimer with a beta 3-specific antibody (B34, 1:150). 1st lane, 300 ng of purified beta 1gamma 5; 2nd lane, 300 ng of purified beta 2gamma 4; 3rd lane, 300 ng of purified beta 3gamma 5; 4th lane, 300 ng of beta gamma purified from Sf9 insect cells.

An example of the purity of the beta 3gamma 4, beta 3gamma 5, and beta 3gamma 11 subunits that can be obtained by this procedure is shown by silver staining. As shown in Fig. 8A, each purified beta 3gamma preparation was composed of two predominant bands by silver staining as follows: a 37-kDa band representing the beta 3 subunit and a 5-8-kDa band representing one of the gamma  subtypes. Confirmation that the beta 3 and gamma 5 subunits interact to form a functional beta gamma dimer is also shown using a previously developed tryptic digestion procedure (19, 27). This method is based on the finding that beta  monomers are cleaved at numerous sites by trypsin. By contrast, functional beta gamma dimers are cleaved at a single site, resulting in the appearance of a 26-kDa fragment of the beta  subunit that is resistant to further digestion by trypsin. Whereas the appearance of this stable 26-kDa fragment is a reliable marker for the formation of beta gamma dimers containing the beta 1 and beta 2 subtypes, it is not clear whether formation of beta gamma dimers containing the beta 3 subtype yields the appearance of a similar protected fragment. To date, such a protected fragment has not been detected for the beta 3 subtype, but these results are difficult to interpret due to the lack of availability of a positive control at that time (19). As shown in Fig. 8B, purified preparations of both the beta 1gamma 5 and beta 3gamma 5 subunits produced a 26-kDa protected fragment when digested under identical conditions with trypsin, as detected in each case by immunoblotting with a commercial beta -antibody (DuPont SW/1, carboxyl terminus). Since equal amounts of beta 1gamma 5 and beta 3gamma 5 subunits were loaded, the differences in intensities of the beta 1 and beta 3 bands presumably reflect differences in affinities of the antibody used for immunoblotting. Taken together, these results confirmed that the beta 3 and gamma 5 subunits are able to interact to form a functional beta gamma dimer. Moreover, these results extended the utility of the trypsin digestion method as a reliable marker of beta gamma dimer formation to the beta 3 subtype.


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Fig. 8.   beta 3gamma dimers purified following expression in Sf9 insect cells. A, silver-stained, purified beta 3gamma dimers. 1st lane, 400 ng of beta 3gamma 5; 2nd lane, 400 ng of beta 3gamma 4; 3rd lane, 400 ng of beta 3gamma 11. B, protection of beta 3 from tryptic proteolysis by association with gamma 5. beta gamma subunits were incubated for 40 min at 30 °C with or without 1 µg of trypsin, after which 6 µg of trypsin inhibitor were added to each, and the samples were trichloroacetic acid-precipitated for 15% SDS-PAGE. The products are shown by Western blot using beta -common antibody (SW/1, NEN Life Science Products). 1st and 2nd lanes, 400 ng each of beta 1gamma 5; 3rd and 4th lanes, 400 ng each of beta 3gamma 5.

Comparison of beta gamma Dimers of Varying beta  Composition in Terms of Coupling to the alpha 2A-Adrenergic Receptor-- As shown in Fig. 9, all combinations of the 6hisalpha i1 subunit with the various beta 3gamma dimers were capable of inducing high affinity [3H]UK-14,304 binding. Again, in all cases, the [3H]UK-14,304 binding was completely abolished by addition of GTPgamma S (data not shown). The beta 1gamma 4 and beta 3gamma 4 dimers showed similar abilities to reconstitute coupling with the recombinant alpha 2A-adrenergic receptor. Likewise, the beta 1gamma 11 and beta 3gamma 11 dimers had essentially identical activities. On the other hand, the beta 3gamma 5 dimer showed a substantially higher level of coupling with the recombinant alpha 2A-adrenergic receptor than the beta 1gamma 5 dimer. Increasing the concentration of the beta 1gamma 5 dimer did not raise the level of coupling (Fig. 5B), indicating the beta 1 subtype, when in association with the gamma 5 subtype, has a lower intrinsic ability to interact with the recombinant alpha 2A-adrenergic receptor. Taken together, these differences support the conclusion that the receptor recognition of the G protein is dependent on the particular combination of beta  and gamma  subtypes.


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Fig. 9.   Comparison of beta 1 and beta 3 in reconstitution of alpha 2A-AR coupling. Gi heterotrimer composed with the beta gamma dimers indicated was reconstituted into Sf9 plasma membranes expressing alpha 2A-adrenergic receptor at a mol/mol ratio of 1:125:100 for receptor:6hisalpha i1:beta gamma . Binding of 4 nM [3H]UK-14,304 was assayed in the absence or presence of 100 µM GTPgamma S. High affinity agonist binding was calculated as the difference between total binding and binding in the presence of GTPgamma S. Background (i.e. high affinity agonist binding without added Gi) was subtracted from each value to show only the reconstituted coupling. beta 1gamma 5 supported significantly less coupling than beta 3gamma 5 (rho  <=  0.05, Student's t test).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The present study examined the potential of the alpha 2A-adrenergic receptor to couple to G proteins differing in their beta gamma subunit composition only. The selectivity of coupling was directly assessed by a high affinity agonist binding assay. Importantly, this assay was found to require the addition of the beta gamma subunits in order to detect the interaction of the alpha  subunit with the receptor. From previous studies, this requirement for the beta gamma subunits appears to reflect not only a general role of the beta gamma subunits to stabilize the alpha  subunit (31) but also a specific role of the beta gamma subunits to interact directly with the receptor (6, 7). In view of these roles and the rich diversity of beta gamma subunit combinations, the possibility was suggested that the nature of the beta gamma subunits might contribute to the selectivity of the receptor interaction. Supporting such a possibility, the present study showed clear differences in the abilities of the various beta gamma dimers, including those containing the beta 3 subtype and the newly described gamma 4, gamma 10, and gamma 11 subtypes, to promote interaction of the same alpha i subunit with the alpha 2A-adrenergic receptor.

Influence of beta gamma Composition on Receptor Coupling-- Several lines of evidence support the validity of using Sf9 insect cell membranes expressing the recombinant alpha 2A-adrenergic receptor as a suitable system for examining the specificity of coupling to purified, recombinant G proteins. First, the recombinant alpha 2A-adrenergic receptor displayed the binding affinities and pharmacologic properties characteristic of the native receptor. Second, the recombinant alpha 2A-adrenergic receptor showed a mostly uncoupled phenotype in the absence of added G proteins and a largely coupled phenotype in the presence of added G proteins of defined composition and stoichiometry. Using high affinity agonist binding as a quantitative measure of the coupled phenotype, this system was first used to examine the influence of the gamma  component on receptor coupling. Gi proteins were produced from 6hisalpha i1, beta 1, and varying gamma  subtypes. Among the eight beta gamma dimers tested, 30-fold differences were observed in their abilities to support coupling of the 6hisalpha i1 subunit to the alpha 2A-adrenergic receptor, with the beta 1gamma 2, beta 1gamma 3, beta 1gamma 4, beta 1gamma 7, and beta 1gamma 11 dimers displaying the most efficacy, the beta 1gamma 5 and beta 1gamma 10 dimers showing intermediate efficacies, and the beta 1gamma 1 dimer exhibiting the least efficacy. With the exception of the gamma 11 subtype, the observed differences segregated with the structural diversity of the gamma  component along subclass lines. As defined previously, the human gamma  subunit family has been divided into three subclasses, with each subclass showing less than 50% amino acid homology to other subclasses (1). On this basis, subclass I contains the gamma 1, gamma c, and gamma 11 subtypes, which are modified by the less common C-15 farnesyl group; subclass II includes the gamma 2, gamma 3, gamma 4, gamma 7, and gamma 12 subtypes, which are modified by the more common C-20 geranylgeranyl group; and subclass III contains the gamma 5 and gamma 10 subtypes, which again receive the more common C-20 geranylgeranyl group. The present study represents the most extensive functional analysis of the gamma  subunit family to date.

Next, this system was used to examine the influence of the beta 1 versus the beta 3 subtype on receptor coupling. In vitro studies have revealed little or no functional differences due to the beta  subunit (16, 17). Since only the closely related beta 1 and beta 2 subtypes were examined, however, the present study extended this analysis to the more divergent beta 3 subtype. For this purpose, the method of Kozasa and Gilman (11) was used to produce and then purify functional beta gamma dimers containing the beta 3 subtype. In addition to providing a source of material of defined composition, this approach also revealed new information on the selectivity of beta -gamma interaction by confirming the ability of the beta 3 subtype to interact with the gamma 4, gamma 5, and gamma 11 subtypes. Whereas interaction between the beta 3 and gamma 4 subtypes had been predicted (2, 3), the ability of the beta 3 subtype to interact with the gamma 11 subtype was unexpected in view of the high homology between the gamma 11 and gamma 1 subtypes and the reported failure of the gamma 1 subtype to interact with the beta 3 subtype (28). When the various beta gamma dimers were tested for receptor coupling, only the beta 3gamma 5 and beta 1gamma 5 dimers showed substantive differences in their abilities to support coupling of the 6hisalpha i1 subunit to the alpha 2A-adrenergic receptor. No such differences were observed with the beta 3gamma 4 and beta 1gamma 4 dimers nor the beta 3gamma 11 and beta 1gamma 11 dimers. These results suggested that it is the particular combination of beta  and gamma  subtypes that ultimately determines receptor recognition. Interestingly, the beta 3gamma 5 dimer was shown previously to interact preferentially with a G protein-coupled receptor kinase, GRK3, indicating a role of the beta  subtype in selective receptor desensitization (32).

Taken together, these data show that Gi proteins containing different beta gamma dimers produce different levels of coupling to the alpha 2A-adrenergic receptor. This result could arise because the composition of the beta gamma subunits alters the formation or stability of the G protein, the affinity of the beta gamma dimer for the receptor, or some combination therefrom. Our data (17) and those from other laboratories (9, 33) suggest that formation of the G protein is the least likely reason since the affinity of the alpha  subunit for the various beta gamma dimers is similar. Instead, our data are most consistent with the composition of the beta  and, particularly, the gamma  component affecting the affinity of the G protein for the alpha 2A-adrenergic receptor. Studies of the A1-adenosine receptor support a similar conclusion (33).

Sites of Interaction of gamma  Component with Receptor-- The observed differences in the abilities of various beta gamma dimers to support coupling to the alpha 2A-adrenergic receptor reside primarily in the gamma  component. Although cross-linking studies have yet to detect receptor-gamma contact sites (7), several studies point to the importance of the carboxyl-terminal amino acid region and the type of prenyl group on the gamma  subunit in determining the interaction of the beta gamma dimer with receptor (34, 35). These latter studies may explain the lower reconstitutive activity of the beta 1gamma 1 dimer in the present study. In this regard, the gamma 1 subtype sequence is the most divergent of those determined to date, and its lipid modification is a C-15 farnesyl group rather than the C-20 geranylgeranyl group found on most other gamma  subtypes (1). With regard to the latter, a recent study comparing beta gamma dimers with the two types of prenyl groups showed that the wild type, geranylgeranylated gamma 2 subunit and the mutant, geranylgeranylated gamma 1 subunit had similar abilities to interact with the A1 adenosine receptor (34). By contrast, the wild type, farnesylated gamma 1 subunit and the mutant, farnesylated gamma 2 subunit were much less effective. Whereas these data indicate that type of prenyl group is critical, the primary structure of the gamma  subunit is of equal or greater importance. Underscoring this point, a synthetic peptide derived from the carboxyl-terminal sequence of the gamma 1 subtype was able to stabilize the light-activated state of rhodopsin receptor to a much greater degree when the peptide was farnesylated than not (35). However, the effect was greatly attenuated when the amino acid sequence of the peptide changed by only two amino acid substitutions (F64T and L67S) even though the farnesylated state was preserved. Thus, both the primary structure and the prenylation state of the gamma 1 subtype are likely to contribute to its poor affinity for the alpha 2A-adrenergic receptor in the present study and its contrastingly high affinity for the rhodopsin receptor in previous studies (35). When compared with the beta 1gamma 1 dimer, the higher reconstitutive activity of the beta 1gamma 11 dimer was unexpected since the newly described gamma 11 subtype is farnesylated and has a carboxyl-terminal tail nearly identical to gamma 1 subtype. This result suggests the importance of regions other than the carboxyl-terminal tail of the gamma 11 subunit in promoting its strong interaction with the alpha 2A-adrenergic receptor. By focusing on the few amino acid differences between the gamma 11 and gamma 1 subtypes and making the appropriate mutations, it should be possible to pinpoint other regions of gamma  protein structure that are selectively recognized by the alpha 2A-adrenergic receptor. Given the wide tissue distribution of the gamma 11 subtype (19) in contrast to the restricted expression of the gamma 1 subtype, it is perhaps not so surprising that a receptor other than rhodopsin would exist which prefers the farnesylated gamma 11 subtype in tissues other than the retina.

Finally, the high reconstitutive activities of the beta 1gamma 2, beta 1gamma 3, beta 1gamma 4 or beta 1gamma 7 dimers compared with the intermediate activities of the beta 1gamma 5 and beta 1gamma 10 dimers underscore the importance of primary structure in another way. Since all of the aforementioned gamma  subtypes are modified by the C-20 geranylgeranyl group (19), the observed differences between the two groups must relate to the differences in protein structure. In agreement with this, the gamma 2, gamma 3, gamma 4 and gamma 7 subtypes share a high degree of amino acid homology (66-74%), and predictably, beta gamma dimers containing these gamma  subtypes produce comparably strong levels of coupling of the 6hisalpha i1 subunit to the alpha 2A-adrenergic receptor. By contrast, the gamma 5 and gamma 10 subtypes share only 35-50% identity with the aforementioned group of gamma  subunits (19), and accordingly, beta gamma dimers containing these gamma  subtypes yielded significantly lower levels of coupling in comparison with the mean value calculated from the grouping of beta gamma dimers consisting of the gamma 2, gamma 3, gamma 4, and gamma 7 subtypes (rho  = 0.025 or 0.05, respectively; Student's t test). Taken together, these data indicate that the primary structure, including regions other than the carboxyl-terminal tail, of the gamma  subunit is a most important factor in determining the selectivity of interaction with the alpha 2A-adrenergic receptor. In this instance, the type of prenyl group is a less critical factor.

Sites of Interaction of beta  Component with Receptor-- Although the beta  subunits are highly conserved in their predicted amino acid sequences, the observed difference between the beta 1gamma 5 and beta 3gamma 5 dimers indicates that selective receptor recognition does occur on the basis of the beta  subtype. Cross-linking studies have revealed a site of contact between the 7th or possibly 6th WD-40 repeat of the beta  subunit and a peptide derived from the third intracellular loop of the alpha 2-adrenergic receptor (7). The crystal structure of beta gamma t indicates that residue 303, which lies at the center of this segment, resides on an exposed surface of the beta gamma dimer (36). Thus, this site has the potential to participate in the preferential coupling of the alpha 2A-adrenergic receptor to Gi heterotrimers containing the beta 3gamma 5 dimer over those containing the beta 1gamma 5 dimer. Other possibilities are suggested: 1) regions other than the carboxyl-terminal tail of the beta  subunit may interact with receptor; 2) a concerted interaction between the beta  and gamma  subunits within the G protein binding pocket of the receptor; or 3) some combination therefrom.

Influence of beta gamma Composition on Coupling to Other G Protein-coupled Receptors-- Various beta gamma dimers show a different order of potency depending on the type of receptor (8, 9, 37). It is speculated that protein-protein interactions between the beta gamma subunits and receptors, as well as hydrophobic interactions due to the prenylation state of the gamma  subunit, will be important elements in modeling selective recognition between G protein and receptors. Distinct, yet to be revealed, structural features within the G protein binding regions of receptor subtypes must also be taken into account in such a model. For example, a previous study showed that the 5HT1A receptor interacts similarly with G proteins containing beta 1gamma 2, beta 1gamma 3, or beta 1gamma 5 dimers (8), whereas the present study revealed that the alpha 2A-adrenergic receptor prefers G proteins containing the beta 1gamma 2 or beta 1gamma 3 dimer over that containing the beta 1gamma 5 dimer. Thus, the G protein binding pockets of the alpha 2A-adrenergic receptor and the 5HT1A may possess subtle structural differences that result in either more or less receptor to Galpha beta gamma contact depending on the identity of the gamma  subunit. Active-state receptors may possess discrete elements contacting alpha , beta , and gamma  subunits within the same G protein that complement one another to some degree in order to activate the heterotrimer. The experimental approach used here is quite amenable to manipulating both the receptor and the G protein subunit composition in order to bring together selected elements of signaling proteins.

Summary-- The data in the present study demonstrate the specificity of alpha 2-adrenergic receptor-G protein interactions is affected by the beta gamma dimer composition, with protein-protein interactions forming the basis for the observed differences. These in vitro results complement a growing body of in vivo results demonstrating the beta gamma subunit composition is an important determinant of the specificity of signaling pathways. Strikingly, antisense suppression of the beta 1gamma 3 or beta 3gamma 4 subtypes disrupts coupling between inhibition of a calcium channel and the somatostatin or muscarinic receptors, respectively, in GH3 pituitary cells (2, 3). Similarly, ribozyme suppression of the gamma 7 subtype attenuates coupling between stimulation of adenylylcyclase and the beta -adrenergic receptor in HEK293 cells (4). Although these results could arise from an ordered arrangement of signaling proteins in the cell membrane, the in vitro results presented here implicate receptor-beta gamma "recognition" as an additional mechanism for determining the specificity of signaling. It is expected that a combination of in vitro and in vivo approaches will provide some of the answers needed for construction of a mechanistic model of specificity in G protein-mediated signaling pathways.

    ACKNOWLEDGEMENTS

We thank the following individuals for their sharing of valuable reagents: Drs. H. Kurose and R. J. Lefkowitz for the pVL1392 transfer vector containing alpha 2A-adrenergic receptor cDNA; and Drs. T. Kozasa and A. G. Gilman for the recombinant baculovirus encoding Galpha i1 subunit containing a hexahistidine tag at amino acid 121.

    FOOTNOTES

* This work was supported by the American Heart Association postdoctoral fellowship (to M. R.) and National Institutes of Health Grant GM39867 (to J. D. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Current address: Anesthesiology Dept., Duke University School of Medicine, Durham, NC 27715.

To whom correspondence should be addressed: Henry Hood Research Program, Pennsylvania State University College of Medicine, Weis Center for Research, Danville, PA 17822-2614.

    ABBREVIATIONS

The abbreviations used are: G protein, heterotrimeric guanine nucleotide-binding regulatory protein; alpha 2-AR, alpha 2-adrenergic receptor; GTPgamma S, guanosine-(5'-thio)-triphosphate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]1-propanesulfonic acid; Ni-NTA, nickel-nitrilotriacetic acid; DTT, dithiothreitol; Sf9 cells, Spodoptera frugiperda cells (ATCC CRL 1711); 6hisalpha i1, hexahistidine-tagged alpha i1 subunit of the G proteins; m.o.i., multiplicity of infection.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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