Biochemistry 1991, 30, 3335-3341
3335
Functional Interactions of Recombinant a2 Adrenergic Receptor Subtypes and G Proteins in Reconstituted Phospholipid Vesicles Hitoshi Kurose, John W. Regan,* Marc G. Caron, and Robert J. Lefkowitz* Departments of Biochemistry, Cell Biology, and Medicine, Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710 Received October 5, 1990; Revised Manuscript Received December 10, 1990
ABSTRACT: The functional interaction of the recombinant a2adrenergic receptor subtypes, a2-C10 (the human
platelet a2receptor, equivalent to the a2A subtype) and a2-C4 (an a2receptor subtype cloned from a human kidney cDNA library), with G proteins was characterized in an in vitro reconstitution system. These receptor subtypes were overexpressed in COS-7 cells and were purified to a specific activity of 1.1-3.3 nmol/mg of protein. The G proteins consisted of G, (adenylyl cyclase stimulatory) and members of the inhibitory family, including Gil, Gi2, and Gi3, and Go. The cloned a subunits of these G proteins were overexpressed in Escherichia coli and were purified to homogeneity. Prior to use, G holoproteins were prepared by mixing the a subunits with & subunits that had been purified from bovine brain. Following reconstitution into phospholipid vesicles, both cy2 receptor subtypes could couple to the inhibitory G proteins but not to G,, as assessed by agonist stimulation of GTPase activity. The pharmacological specificity of this interaction was preserved with respect to the two receptor subtypes. Between the different inhibitory G proteins, the a2-C10adrenergic receptor subtype showed the following preference: Gi3> Gil 1 Gi2> Go. The stimulation of GTPase activity (turnover number) ranged from 6.4-fold (Gi3) to 1.5-fold (Go). The preference of G-protein interaction for the a2-C4 receptor subtype was the same as that observed for the a2-C10, but the extent of activation was slightly lower. The results show that in vitro each of the a2adrenergic receptor subtypes can activate multiple G proteins but that clear preferences exist with respect to the individual inhibitory G-protein subtypes. Additionally, it appears that a,-C10 is coupled more efficiently to G-protein activation than is a2-C4.
G u a n i n e nucleotide binding regulatory proteins (G proteins)’ are a family of proteins that serve as intermediates in the transduction of external stimuli across the cell membrane (Gilman, 1987; Lochrie & Simon, 1988). These proteins are composed of three subunits. The a subunit, the largest, binds and hydrolyzes guanine nucleotides and appears to be the most important subunit in terms of giving a G protein its particular characteristics, e.g., stimulatory versus inhibitory. The two other subunits, known as 07, are associated and normally form a complex along with the a subunit. External stimuli, for example, in the form of a hormone-receptor interaction, lead to a further interaction with the G proteins that causes the & and a subunits to dissociate. In most cases, the a subunit appears to be the one that subsequently interacts with downstream effector molecules. The precise role of the & subunits is not clear, although under some conditions they may also regulate certain effector proteins (Jelsema & Axelrod, 1987; Katada et al., 1987; Bourne, 1989; Kim et al., 1989). The DNA’s encoding each of these subunits have been cloned and have revealed considerable heterogeneity, particularly of the a subunits (Gilman, 1987; Lochrie & Simon, 1988). At present, at least 16 species of this subunit have been identified, including a subgroup that are sensitive to pertussis toxin. This bacterial toxin catalyzes ADP ribosylation of a cysteine that inactivates the pertussis toxin sensitive G proteins. Members of this subgroup, including Gi,, Gi2, GI3,and Go, have been studied extensively because of their involvement with a variety of cellular responses, including inhibition of adenylyl cyclase, modulation of ion channels, and activation of phospholipase C (Ui, 1986; Limbird, 1988).
* To whom correspondence should be addressed.
* Present address:
Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, AZ 85721.
As mentioned above, G proteins transduce external stimuli, usually in the form of a hormone-receptor interaction, into a cellular response. Among the receptors that interact with G proteins are the adrenergic receptors. Adrenergic receptors serve as the initial contact in this signal transduction system by binding endogenous catecholamines, which have the capacity to function as either hormones or neurotransmitters. The adrenergic receptors consist of a family of proteins that have been subclassified by physiological, pharmacological, and biochemical means and, more recently, with the cloning of their genes and/or cDNAs (Lefkowitz & Caron, 1988). The basic subclassification consists of the 0and a adrenergic receptors. The a adrenergic receptors are further divided into the a1and a2 subtypes. A suggestion for the classification of the a2 adrenergic receptors into A and B subtypes was made on the basis of pharmacological studies (Bylund, 1985). Thus, the a2A was recognized as having high affinity for oxymetazoline and low affinity for prazosin, whereas this was the opposite for the a2B subtype. Heterogeneity of the a2adrenergic receptors has been confirmed by purification and cloning studies. The human platelet a2 adrenergic receptor, which is the prototypic a2A, has been purified and its gene cloned (Kobilka et al., 1987). By use of the latter as a probe, a novel a2 adrenergic receptor subtype was cloned from a human kidney cDNA library (Regan et al., 1988). Expression of this receptor showed that it had pharmacological characteristics similar to the a2B subtype; however, it has been called an a2-C4 until Abbreviations: G proteins, guanine nucleotide binding regulatory proteins; G,, G protein that mediates stimulation of adenylyl cyclase; Gi, G protein that mediates inhibition of adenylyl cyclase; Go,G protein of unknown function; GTPyS, guanosine S’-(y-O-thio)triphosphate;App(NH)p, adenyl-5’-yl imidodiphosphate; Hepes, 4-(2-hydroxyethyl)-lpiperazineethanesulfonicacid.
0006-2960/91/0430-3335$02.50/0 0 1991 American Chemical Society
Kurose et al.
3336 Biochemistry, Vol. 30, No. 13, 1991 more confirmatory data are obtained. A designation similar to C4, which refers to the human chromosomal localization, has also been applied to the human platelet a2 adrenergic receptor, which in this study is referred to as the a,-C10. The DNA's encoding the a2-C4 and a2-C10 adrenergic receptors have been used to transfect cultured mammalian cells, and stable clones have been obtained (Cotecchia et al., 1989). In these cells, activation of a2-C10 inhibited adenylyl cyclase approximately 70%, whereas maximal inhibition by a2-C4 was only about 30%. Additionally, both receptors could weakly stimulate phosphatidylinositol hydrolysis. Pretreatment of the cells with pertussis toxin blocked both the inhibition of adenylyl cyclase and the effects on phosphatidylinositol turnover. These results implicated pertussis toxin sensitive G protein(s) in the coupling of these signal transduction pathways; however, whether these responses were mediated through a single G protein or through multiple G proteins could not be assessed. For example, it could not be determined whether a,-C10 and a2-C4were activating unique G proteins that were coupled to the same response, whether they activated a common G protein that was coupled to multiple responses, or whether they activated common G proteins that were each coupled to a unique response. Because of the number of potential players and the complexity of intact cell systems, the best way to examine the potential for these interactions is with a reconstituted system using the purified components. Accordingly, using recombinant DNA techniques, we have overexpressed two a2receptor subtypes and five G-protein subtypes in COS-7 cells and in Escherichia coli, respectively. These proteins have been purified, and their interactions have been examined following reconstitution into phospholipid vesicles. It is apparent that both receptor subtypes have the potential to activate multiple signal transduction pathways by activating multiple G proteins. EXPERIMENTAL PROCEDURES Materials. [3H]Yohimbine (70.5-88.4 Ci/mmol) and [35S]GTPyS(1208-1 350 Ci/mmol) were purchased from Du Pont New England Nuclear. Epinephrine, octyl fl-D-glucopyranoside (octyl glucoside), soybean phosphatidylcholine (type 11-S), GTP, and App(NH)p were obtained from Sigma; [y32P]GTP (650 Ci/mmol) was from ICN, GTPyS was from Boehringer Mannheim, and digitonin was from GallardShlesinger. Sources of drugs were as follows: yohimbine, Aldrich; rauwolscine, Roth, Karlsruhe, FRG; prazosin, Pfizer; and phentolamine, Ciba-Geigy. Expression and Purification of a2Receptors and G Proteins. a,-C10 and a2-C4 were expressed in COS-7 cells by use of the vectors and transfection procedures previously described by Regan et al. (1988) with the following modification. To increase the expression of pBCa2-C4, a new construct was made in which all of the 5' untranslated region and most of the 3' untranslated region were removed. In this vector, the a2-C4 coding sequence (Regan et al., 1988) was placed inframe with the vector initiator methionine of pBC12RF (Cullen, 1987) such that the final product contains six additional amino acids (MALWIP) from the human pre-proinsulin gene. This vector (pBCa2-C4-#6) contains the NcoISmaI fragment of az-C4 (nucleotides -0-1460) inserted into pBC12RF from which the SmaI fragment (nucleotides 506-1 207) was removed. In this study, the level of expression of cy2-C10 and az-C4 was 10-1 5 and 3-5 pmol/mg of protein, respectively. The a2-C10and a2-C4adrenergic receptors were solubilized from COS-7 cell membranes with digitonin and were partially purified by affinity chromatography followed by heparin-
-
agarose chromatography to a specific activity of 1.l-3.3 nmol/mg of protein. The purity of the receptor preparations was 6-20% based on a molecular weight of 67 000 or 75 000 in COS-7 cell membranes for a2-C10 or a2-C4, respectively (Regan et al., 1988). COS-7 cells were harvested into a cold buffer containing 20 mM Hepes (pH KO), 2 mM EDTA, and 0.1 mM PMSF and homogenized with a Brinkman PT 10/35 Polytron. After centrifugation at 43000g for 20 min, the pellet was frozen in liquid Nz and stored at -80 OC until use. For solubilization, the pellet was thawed and centrifuged at 43000g for 20 min. The pellet was homogenized for 3 min in the solubilization buffer at a concentration of 3 mg/mL with Brinkman PT 10/35 Polytron. The solubilization buffer contained 20 mM Hepes (pH 8.0), 5 mM EDTA, 2 mM EGTA, 0.1 mM PMSF, and 1% digitonin. The suspension was stirred overnight (approximately 16 h) in a cold room and centrifuged at 43000g for 60 min. The supernatant was immediately used as solubilized receptors. Partial purification of the receptors was the same procedure as that described by Regan and Matsui (1990). The a2adrenergic receptors thus purified were stored at 4 "C in a buffer containing 50 mM Tris (pH 7.2), 1 mM EDTA, 1 mM dithiothreitol, 100 pM phentolamine, 500-700 mM NaCI, and 0.05% digitonin. Recombinant G-protein a subunits, which were purified after expression in E. coli, were provided by Drs. Linder and Gilman (Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75235). Details of the purification and characterization of these proteins are described elsewhere (Graziano et al., 1989; Linder et al., 1990). These G-protein a subunits were more than 95% pure based on a Coomassie Blue staining of the gel, and the preparations of subunits were 5040% active as assessed by [35S]GTPyS binding (Linder et al., 1990). The j3y subunits were purified from bovine brain (Sternweis & Robishaw, 1984; Casey et al., 1989). The amount of contaminating GTP-binding activity in the fly preparation was 0.25%. Reconstitution of Receptors and G Proteins into Phospholipid Vesicles. Reconstitution was performed according to the method of Florio and Sternweis (1985) with the following modifications. Receptors (2.5 pmol of a,-C10 or 2.5-4.0 pmol of a2-C4 in digitonin solution), a subunits of G proteins (5-8 pmol for ai,, ai2,and ai3or 11-18 pmol for cyo, except Figure 3), fly subunits ( N 10 pmol for ai,, ai2,and ai3 or -70 pmol for ao,except Figure 3), soybean phosphatidylcholine (250 pg sonicated in 0.4% octyl glucoside solution), phentolamine (final concentration 100 pM), and octyl glucoside (final concentration 0.2%) were combined in a total volume of 500 p L by use of 20 mM Hepes (pH 8.0)/1 mM EDTA/160 mM NaCl to adjust the volume. Reconstitution was effected by gel filtration over a column of Sephadex G-50 (0.6 X 12.5 cm, 3.5-mL bed volume) that was preequilibrated and eluted with the previous buffer. After the first 650 pL of the column elution was discarded, the next 1 mL was collected as reconstituted vesicles and used directly for the assay. GTP Hydrolyzing (GTPase) Activity. GTPase activity was assayed by incubation of reconstituted vesicles at 30 OC in a total volume of 100 pL of assay buffer containing an additional 0.1 p M [T-~~P]GTP (18000&320000 cpm/pmol) and 10 mM MgC1,. The assay buffer consisted of 20 mM Hepes (pH 8.0), 1 mM EDTA, 160 mM NaC1,0.2 mM App(NH)p, 1% bovine serum albumin, and 0.2 mM ascorbic acid. The incubation time is shown in the figures. The reaction was stopped by the addition of 10 p L of 50% trichloroacetic acid. [32P]Pirelease was measured as described by Cerione et al. (1985). Sig-
Recombinant a2 Receptor/G Protein Reconstitution nificant GTPase activity was absent in control vesicles containing a2 adrenergic receptors alone. [35S]GTPySBinding. The binding of [35S]GTPyS was measured as described by Cerione et al. (1985) and Senogles et al. (1990). Vesicles were incubated at 30 "C in assay buffer supplemented with 10 mM MgC12 and 0.1 pM [35S]GTPyS (140000-260000 cpm/pmol). The incubation time is shown in the figures. Assays were stopped by the addition of 400 pL of 0.5% cholate solution. Nucleotide bound to G protein was separated by gel filtration on Sephadex G-50 (0.6 X 12.5 cm, 3.5-mL bed volume) that was preequilibrated and eluted with a buffer containing 20 mM Tris (pH 7.4), 100 mM NaCl, 25 mM MgCl,, and 0.1% cholate. Nonspecific binding was determined in the presence of 10 pM GTPyS. The total amount of G protein present in the vesicles was estimated by the amount of [35S]GTPySbound in the presence of 20 mM MgC12,0.05% Lubrol, and 0.1 pM [35S]GTPySafter 60 min of incubation at 30 OC. ['HI Yohimbine Binding. Vesicles containing receptors were assayed with 10.0 nM [3H]yohimbineto determine the total binding activity. All binding assays were performed in 50 mM Tris (pH 7.2)/1 mM EDTA/O.l% digitonin at 4 OC overnight. The free and bound [3H]y~himbinewere separated by gel filtration on Sephadex G-50 (0.6 X 12.5 cm, 3.5-mL bed volume) that was preequilibrated and eluted with 50 mM Tris (pH 7.2)/ 1 mM EDTA/O. 1% digitonin. Nonspecific binding was determined in the presence of 30 pM phentolamine. RESULTS Functional Reconstitution of a2-ClO and a,-C4 Adrenergic Receptor Subtypes with G Proteins. Recombinant DNA encoding the a2-C10 and a2-C4 adrenergic receptor subtypes was used to transfect COS-7 cells, and the expressed receptors were purified to a specific activity above 1.0 nmol/mg of protein. Reconstituted vesicles were obtained by gel filtration over Sephadex (3-50 because of its speed and high recovery of binding activity ( 2 5 4 0 % for a2-C10 and 15-30% for a2-C4). For unknown reasons, the reconstitution efficiency of a2-C4 was always about half of that of a2-C10 and there was no consistent tendency of the receptors to incorporate into the phospholipid vesicles with different amounts of each G protein. Recovery of G proteins into phospholipid vesicles was 30-55%, 20-40%, 20-4056, or 15-35% for Gil, Gi2,Go, or Go, respectively, and was independent of the a2receptor subtype that was used in the reconstitution. Functional interactions of the a2receptor subtypes with the G proteins were monitored by measuring GTPase activity and [35S]GTPySbinding. The time courses of this activity and binding are shown in Figure 1 following reconstitution of a,-C10 with vesicles containing Gi3. GTPase activity was linear up to 25 min and was stimulated up to 8-fold by the addition of the agonist epinephrine (Figure 1A). For experimental purposes, an incubation time of 20 min was chosen for determining the interaction of the two receptor subtypes with the various G proteins. The reason for this was primarily because of the variability associated with the low counts per minute at the earlier time points. Figure 2 shows the ability of various antagonists to block epinephrine-stimulated GTPase activity for the two receptor subtypes. a2-C10 and a2-C4were each reconstituted with Gi3, and GTPase activity was determined following stimulation with 5 pM epinephrine in the presence of increasing concentrations of rauwolsine, yohimbine, or prazosin. For a,-C10, the order of potency was rauwolsine = yohimbine >> prazosin (Figure 2A). For a2-C4, the order of potency was rauwolsine > yohimbine > prazosin (Figure 2B). These findings are in good agreement with the results obtained from competition binding
Biochemistry, Vol. 30, No. 13, 1991 B
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t
i; 1.0 5
1
300
20
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Time (mln)
FIGURE 1 : Agonist-stimulated GTPase activity and [%]GTPyS binding to reconstituted vesicles. The a2-C10/Gi3vesicles were prepared as described under Experimental Procedures. (A) Vesicles were incubated at 30 OC in an assay mixture containing 10 mM MgCI2 and 0.1 pM [32P]GTPand in the presence ( 0 )or absence (0)of 10 pM epinephrine. At the indicated times, aliquots were withdrawn into stop solution and were assayed for released [32P]Pi.(B) Vesicles containing az-CIOand Go were incubated at 30 OC in an assay mixture with the addition of 10 mM MgC12 and without (0)or with (0)10 pM epinephrine. At the indicated times, aliquots were withdrawn into 0.5% cholate solution and were assayed for [35S]GTPySbinding. The data shown are representative of two experiments.
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FIGURE 2: GTPase activity of reconstituted vesicles containing a2-C10 or az-C4 and Gi3. Vesicles containing a2-C10 (A) or az-C4 (B) and Gi3were prepared as described under Experimental Procedures. The reconstituted vesicles were incubated at 30 OC for 20 min in an assay mixture containing 10 mM MgClz and 0.1 pM [32P]GTPand increasing concentrations of yohimbine (0),rauwolsine (A),or prazosin ( 0 ) . GTPase activity was normalized to the activity without antagonist. The EC5 Rb+ > NH4+ > Cs+ >> Na+, Li+. The currents are inhibited by Ba2+, Cs', and tetraethylammonium (TEA), common pore blockers of K+ channels. Open-channel blockade by Cs+ (but not by Ba2+ or TEA) depends on applied voltage. The major inhibitory effect of Ba2+ is to alter channel gating by favoring the closed state; this effect is specific for Ba2+ and is relieved by external K+. The results argue that although the polypeptide expressed is very small for a eukaryotic ion channel, 130 amino acid residues in length, the ionic currents observed are indeed mediated by a genuine K+-channel protein. This synthetic gene is therefore well suited for a molecular analysis of the basic mechanisms of K+-channel function. ABSTRACT:
&age-dependent K+ channels are centrally involved in the generation of electric signals in excitable cell membranes (Hille, 1984). In order to fulfill their physiological duties, channels of this class must carry out two essential tasks. First, in response to changes in membrane voltage, the channel protein must alter its conformation to form a water-filled transmembrane pore through which ions can diffuse passively. Second, the open pore thus formed must discriminate strongly among inorganic ions, allowing rapid permeation for K+ but preventing Na+ from traversing the pore. Very little is known about the molecular mechanisms by which these tasks are accomplished; only in the last few years have cDNA clones for K+ channels become available (Papazian et a]., 1987; Pongs et al., 1988; Kamb et al., 1988; Frech et al., 1989; Stuhmer et al., 1989; Butler et a]., 1990), and work on structurefunction relations through site-directed mutagenesis is only now beginning (Hille, 1984; Ruppersberg et al., 1990; MacKinnon & Miller, 1989; Isacoff et al., 1990; MacKinnon & Yellen, 1990; Hoshi et al., 1990). Nearly all K+ channels for which genes have been identified belong to a single molecular family consisting of rather large ( ~ 7 0 - k D a )polypeptides that span the membrane at least six times. The
functional channel is believed to be formed as a tetramer of these 70-kDa subunits (Catterall, 1988; Jan & Jan, 1989; MacKinnon, 1991). Recently, several groups employing heterologous expression in Xenopus oocytes cloned a gene putatively coding for a voltage-dependent K+ channel from mammalian kidney, uterus, and heart (Takumi et al., 1988; Murai et al., 1989; Folander et al., 1990; Pragnell et al., 1990). The molecular characteristics of this cDNA are unprecedented among all other known K+ channels. The gene codes for a very small (- 15-kDa) polypeptide of only 130 amino acids. Moreover, hydropathy analysis of this polypeptide identifies only a single membrane-spanning a-helix (Takumi et al., 1988). These remarkable properties have prompted workers in the field to name this channel "minK" (minimal K). There are currently two points of fundamental uncertainty about the nature of this unusual clone. First, it is still unclear whether the K+ currents induced in oocytes by expression of this cDNA are in fact mediated by a channel-type K+ transporter; since direct observations of single channels underlying this K+ current have not yet been achieved, it is still possible that a "carrier-type" mechanism is responsible.
0006-296019 110430-3341 ~$02.50 - .., ~IO. - 0 1991 American Chemical Society I
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