Characterization of the Extra-large G Protein α-Subunit XLαs

In the preceding paper (Pasolli, H. A., Klemke, M., Kehlenbach, R. H., Wang, Y., and Huttner, W. B. (2000) J. Biol. Chem. 275, 33622–33632), we report on the tissue distribution and subcellular localization of XLαs (extra large αs), a neuroendocrine-specific, plasma membrane-associated protein consisting of a novel 37-kDa XL domain followed by a 41-kDa αs domain encoded by exons 2-13 of the Gαs gene. Here, we have studied the signal transduction properties of XLαs. Like Gαs, XLαs undergoes a conformational change upon binding of GTPγS (guanosine 5′-O-(thio)triphosphate), as revealed by its partial resistance to tryptic digestion, which generated the same fragments as in the case of Gαs. Two approaches were used to analyze XLαs-βγ interactions: (i) ADP-ribosylation by cholera toxin to detect even weak or transient XLαs-βγ interactions and (ii) sucrose density gradient centrifugation to reveal stable heterotrimer formation. The addition of βγ subunits resulted in an increased ADP-ribosylation of XLαs as well as an increased sedimentation rate of XLαs in sucrose density gradients, indicating that XLαs interacts with the βγ dimer. Surprisingly, however, XLαs, in contrast to Gαs, was not activated by the β2-adrenergic receptor upon reconstitution of S49cyc− membranes. Similarly, using photoaffinity labeling of pituitary membranes with azidoanilide-GTP, XLαs was not activated upon stimulation of pituitary adenylyl cyclase-activating polypeptide (PACAP) receptors or other Gαs-coupled receptors known to be present in these membranes, whereas Gαs was. Despite the apparent inability of XLαs to undergo receptor-mediated activation, XLαs-GTPγS markedly stimulated adenylyl cyclase in S49cyc− membranes. Moreover, transfection of PC12 cells with a GTPase-deficient mutant of XLαs, XLαs-Q548L, resulted in a massive increase in adenylyl cyclase activity. Our results suggest that in neuroendocrine cells, the two related G proteins, Gαs and XLαs, exhibit distinct properties with regard to receptor-mediated activation but converge onto the same effector system, adenylyl cyclase.

In the preceding paper (32), we show that XL␣s (for extra large ␣s), an unusual type of G protein ␣ subunit, is predominantly associated with the plasma membrane of certain neuroendocrine cells. XL␣s consists of a novel 37-kDa XL domain followed by a 41-kDa ␣s domain encoded by exons 2-13 of the G␣s gene (1,2) and, hence, contains most of the functional domains of G␣s including receptor and effector binding sites. Given the subcellular localization of XL␣s and its domain structure and in light of the observation that the C-terminal sequence of the XL domain shows a high homology to the exon 1-encoded portion of G␣s (1), which promotes binding to the ␤␥ complex, it is important to determine whether or not XL␣s functions, like G␣s, in signal transduction.
In the present study, we addressed the following questions. First, does XL␣s exchange guanine nucleotides and, if so, does this guanine nucleotide exchange lead to a conformational change of XL␣s, as has been reported for G␣s (3)? Second, does XL␣s interact with the ␤␥ complex? Third, does XL␣s couple to heptahelical receptors and, if so, to which ones? And, finally, does XL␣s activate adenylyl cyclase, the classical G␣s effector?

Antibodies
The rabbit antiserum RK5 (anti-XL) against the EPAA-repeats in the XL domain of XL␣s was that described in the preceding paper (32). The rabbit antiserum against the C-terminal decapeptide of G␣s and XL␣s (anti-␣s C terminus) was the same as described previously (1).

Plasmids
The plasmid CDM8-XL␣s, originally called CDM8-XL, contains a 2.6-kilobase insert starting at nucleotide 380 of the originally published sequence (1) and encodes the entire XL␣s protein sequence (see correction of translational start (2)) under the control of the cytomegalovirus promotor.
For construction of CDM8-XL␣s-wt, the plasmid pGEM-G␣s (kindly provided by Dr. Peter Gierschik, University of Ulm), that encodes the entire rat G␣s protein sequence, was cut with Eco47III and NsiI. The resulting 667-nucleotide fragment, corresponding to amino acid residues 165-386 of G␣s, was cloned into the Eco47III and NsiI sites of CDM8-XL␣s, resulting in a predicted XL␣s protein sequence containing a leucine at position 519 (corrected translational start (2)) instead of a proline as in CDM8-XL␣s (amino acid residue 650 of originally published sequence (1)). The nucleotide exchange and the ligation sites were confirmed by sequencing.
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s)AF116268.
§ Recipient of a fellowship from Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina. Present address: Centro de Microscopia Electronica, Universidad Nacional de Cordoba, C. Correo 362, 5000-Cordoba, Argentina. For construction of CDM8-XL␣s-Q548L, the plasmid pVL-1393-G␣s-Q227L (kindly provided by Dr. Peter Gierschik, University of Ulm), which encodes the entire human G␣s protein sequence, was cut with Eco47III and NsiI. The resulting 667-nucleotide fragment, corresponding to amino acid residues 165-386 of G␣s, was cloned into the Eco47III and NsiI sites of CDM8-XL␣s, resulting in a predicted XL␣s protein sequence carrying a single point mutation (Gln 3 Leu) at amino acid residue 548 (corrected translational start (2); amino acid residue 679 of originally published sequence (1). The other differences in nucleotide sequence between human pVL-1393-G␣s-Q227L and the rat CDM8-XL␣s do not cause any amino acid sequence variation between the two species.) The nucleotide exchange and the ligation sites were confirmed by sequencing. The plasmid CDM8-G␣s (kindly provided by Yanzhuang Wang of our laboratory) encodes the entire rat G␣s protein sequence under the control of the cytomegalovirus promotor.

In Vitro Transcription
After linearization by NdeI, 10 g of each plasmid (CDM8-G␣s and CDM8-XL␣s-wt) was in vitro transcribed for 4 h at 37°C in a final volume of 100 l containing 20 l of 5ϫ transcription buffer (MBI Fermentas), 3 l each of ATP, GTP, CTP, and UTP (100 mM each), 2 l of RNase Inhibitor (40 units/l), 3 l of T7 RNA polymerase (40 units/ l), and nuclease-free distilled H 2 O. Two h after the addition of the T7 RNA polymerase, another 3 l of the polymerase were added. Two l of a 1:10 dilution of the total in vitro transcription mixture were used directly for in vitro translation.

In Vitro Translation
Cell-free translation of in vitro transcribed RNAs was carried out at 30°C for 1 h using the Promega nuclease-treated reticulocyte lysate following the manufacturer's instructions. Briefly, a typical translation mixture contained 35 l of the reticulocyte lysate, 7 l of nuclease-free distilled H 2 O, 1 l of RNase Inhibitor (40 units/l), 1 l of the amino acid mixture without methionine, 4 l of the L-[ 35 S]Met/Cys ProMix TM (Amersham Pharmacia Biotech, 1000 Ci mmol Ϫ1 ), and 2 l of the 1:10 diluted total in vitro transcription mixture containing the RNA template. The non-radioactive in vitro translation for ADP-ribosylation and the reconstitution of S49cyc Ϫ membranes was performed with 1 l of the amino acid mixture without methionine and 1 l of the amino acid mixture without cysteine.

Immunoprecipitation
All steps were performed at 4°C. In vitro translated G␣s and XL␣s were mixed with two volumes of immunoprecipitation buffer (3% Triton X-100, 1.5% sodium deoxycholate, 0.3% SDS, 450 mM NaCl, 3 mM EDTA, 3.75 mM phenylmethylsulfonyl fluoride (PMSF), 1 and 30 mM Tris-Cl, pH 8.0) and incubated for 30 min. Insoluble material was removed by centrifugation for 20 min at 14,000 ϫ g, and the supernatant was incubated for 30 min with 50 l of a 50% slurry of protein A-Sepharose and centrifuged for 5 min at 800 ϫ g. The supernatant was used for immunoprecipitation using the antiserum against the common C-terminal decapeptide of G␣s and XL␣s or the anti-XL antiserum. The samples were incubated with the antibody overnight followed by the addition of protein A-Sepharose (50 l of a 50% slurry) and further incubation for 2 h. The Sepharose beads were pelleted and washed twice with buffer A (0.2% Triton X-100, 150 mM NaCl, 2 mM EDTA, 10 mM Tris-Cl, pH 8.0) and buffer B (0.2% Triton X-100, 450 mM NaCl, 2 mM EDTA, 10 mM Tris-Cl, pH 8.0) and once with buffer C (0.1% Triton X-100, 10 mM Tris-Cl, pH 8.0). Immunoprecipitated material was analyzed by SDS-PAGE and phosphoimaging.

Tryptic Digestion
Tryptic digestion of G␣s and XL␣s was performed as described previously (4). Briefly, in vitro translated 35 S-labeled proteins were incubated for 10 min at 37°C in TMED (25 mM Tris-Cl, pH 8.0, 10 mM MgCl 2 , 1 mM EDTA, 1 mM DTT) in the absence or presence of 100 M GTP␥S and then digested for 1 h at 30°C in the presence of various concentrations (0 -0.5 g/l) of trypsin (as L-1-tosylamido-2-phenyl-ethyl chloromethyl ketone-treated; Sigma). Digestion was stopped by the addition of SDS sample buffer immediately followed by boiling of the samples for 5 min at 95°C.

Cholera Toxin-catalyzed ADP-ribosylation
ADP-ribosylation of in vitro translated XL␣s was performed by a modification of the procedure of Audigier (3). In vitro translated ␣-subunit (20 l) was mixed with 30 l of 20 mM HEPES-KOH, pH 7.2, 2 mM MgCl 2 , and 1 mM EDTA. After 15 min of incubation on ice, 0.5 l of buffer (20 mM Tris-Cl, pH 8.0, 2 mM MgCl 2 , 1 mM EDTA, 2 mM DTT, and 11 mM CHAPS) either lacking or containing 43 ng ␤ 1 ␥ 2 was added to the mixture, followed by the immediate addition of 17.5 l of 0.5 M Na 3 PO 4 , pH 7.2, 60 mM thymidine, 5 mM ATP, 0.5 mM GTP, 5 mM MgCl 2 , and 10 Ci of 32 P-NAD ϩ (800 Ci mmol Ϫ1 , PerkinElmer Life Sciences). Cholera toxin-catalyzed ADP-ribosylation was initiated by the addition of 10 l of a 0.5 mg/ml solution of cholera toxin A-subunit activated with 25 mM dithiothreitol for 30 min at 37°C before use. After 60 min of incubation at 30°C, the reaction mixture was transferred on ice, and 4 volumes of immunoprecipitation buffer (100 mM Tris-HCl, pH 7.5, 100 mM KCl, 5 mM MgCl 2 , 1% Triton X-100, 1% sodium deoxycholate, 0.5% SDS, and 1 mM PMSF) were added. Insoluble material was removed by centrifugation for 15 min at 14,000 ϫ g at 4°C. The supernatant was incubated overnight with 20 l of the rabbit antiserum against the C-terminal decapeptide of G␣s and XL␣s. Immune complexes were collected using protein A-Sepharose in PBS and analyzed by SDS-PAGE and autoradiography.

Sedimentation Analysis
Sedimentation analysis using sucrose density gradient centrifugation was performed as described previously, with minor changes (5). Briefly, 5 l of G␣s or XL␣s translation medium was incubated for 24 h at 0°C in the absence or presence of 150 ng of purified unlabeled ␤ 1 ␥ 2 subunits (kindly provided by Dr. Christiane Kleuss, Free University of Berlin) in a buffer containing 50 mM Tris-Cl, pH 8.0, 10 mM EDTA, 1 mM DTT, and 1 mM GDP␤S in a final volume of 100 l. Samples are loaded on top of linear 5-30% sucrose density gradients prepared from solutions also containing 20 mM Tris-Cl, pH 8.0, 1 mM EDTA, and 10 mM ␤-mercaptoethanol. Gradients were centrifuged for 18 h in a Beckman SW60 rotor at 55,000 rpm at 4°C with the deceleration setting "slow," and 20 fractions were collected per gradient.

Cell Culture and Transfection
PC12 cells were plated on 15-cm dishes and were grown to subconfluency in Dulbecco's modified Eagle's medium supplemented with 10% horse serum and 5% fetal calf serum at 10% CO 2 and 37°C. For transient transfection, cells harvested from a subconfluent 15-cm dish after trypsinization were subjected to electroporation (Bio-Rad Gene Pulser; 960 F, 300 V) in 0.8 ml of phosphate-buffered saline containing 45 g of circular plasmid DNA. Transfected cells were plated on a 15-cm dish and used 2 days after transfection, with 10 mM sodium butyrate added during the last 16 h to increase the expression of the transgene (6). S49cyc Ϫ cells were grown in flasks to a density of 1 ϫ 10 5 -2 ϫ 10 6 cells/ml in Dulbecco's modified Eagle's medium (4.5 mg/ml glucose) supplemented with 10% fetal calf serum at 5% CO 2 and 37°C.

Membrane Preparations
PC12 Membranes-A post-nuclear supernatant (PNS) from PC12 cells was prepared as described (7). For the determination of adenylyl cyclase activity, total membranes were prepared from the PNS by centrifugation (1 h, 100,000 ϫ g, 4°C), resuspended in 20 mM HEPES-KOH, pH 7.2, at 1-2 mg of protein/ml, and snap-frozen in liquid nitrogen.
S49cyc Ϫ Membranes-S49cyc Ϫ cells (50 ml, ϳ10 6 cells/ml) were pelleted for 5 min at 800 rpm in a Heraeus Megafuge at 4°C and washed once in ice-cold phosphate-buffered saline containing 0.5 mM PMSF. The cells were resuspended in 10 ml of homogenization buffer (0.25 M sucrose, 1 mM EDTA, 1 mM magnesium acetate, 1 mM DTT, 0.5 mM PMSF, and 10 mM HEPES-KOH, pH 7.4) and pelleted for 5 min at 1600 rpm in a Heraeus Megafuge. The cells were resuspended in 800 l of homogenization buffer and homogenized by passage through a 22-gauge needle attached to a 1-ml syringe followed by 10 passes through a cell cracker (EMBL, 12-m clearance). The homogenate was centrifuged for 10 min at 900 ϫ g at 4°C, and the resulting PNS was centrifuged for 1 h in a Beckman TLA45 rotor at 43,000 rpm at 4°C. The membrane pellet was resuspended in 10 mM HEPES-KOH, pH 7.4, 1 mM DTT to a final protein concentration of 2 mg/ml, snap-frozen in liquid nitrogen, and stored at Ϫ80°C. Rat Pituitary Membranes-Adult rats (Wistar strain) were anesthetized with chloroform and killed by cervical dislocation. The pituitaries were removed from the skull and transferred into ice-cold HBS (0.3 M sucrose, 1 mM MgCl 2 , 1 mM EDTA, 1 mM PMSF, and 10 mM HEPES-KOH, pH 7.4). The pituitaries were homogenized in 5 volumes of HBS using a motor-driven glass-Teflon homogenizer at 1000 rpm. The homogenate was centrifuged for 10 min at 1000 ϫ g at 4°C. The resulting PNS was centrifuged for 1 h at 100,000 ϫ g at 4°C. The membrane pellet was resuspended to a protein concentration of 2 mg/ml in 10 mM Tris-Cl, pH 7.4, snap-frozen in liquid nitrogen, and stored at Ϫ80°C.

Reconstitution of S49cyc Ϫ Membranes with in Vitro Translated G␣s and XL␣s
Reconstitution of the S49cyc Ϫ membranes was performed as described previously (8). Briefly, 1 volume of the in vitro translation mixture was mixed with 1 volume of S49cyc Ϫ membranes (2 mg of protein/ml) and incubated for 30 min at 30°C, followed by centrifugation for 30 min in a Beckman TLA45 rotor at 43,000 rpm at 4°C. The resulting membrane pellet was resuspended to a protein concentration of 1 mg/ml in 10 mM Hepes, pH 7.4, 1 mM DTT. The reconstituted membranes were kept on ice and immediately used for the adenylyl cyclase assay.

Adenylyl Cyclase Assay
The activity of adenylyl cyclase was determined by the method of Salomon (9)  Photoaffinity Labeling Using Pituitary Membranes-AA-GTP labeling was performed essentially as described previously (10). Briefly, membranes (100 g of protein) were pelleted (10 min, 14,000 ϫ g) and resuspended in 30 l of incubation buffer (0.2 mM EDTA, 10 mM MgCl 2 , 60 mM NaCl, 100 mM HEPES-KOH, pH 7.5, 2 mM benzamidine, and 2-200 M GDP). Samples then received 10 l without or with the indicated receptor agonists (see text under "Results" and the legend to Fig. 8), were incubated for 3 min at 30°C, received 20 l of [␣-32 P]AA-GTP (1.4 ϫ 10 6 cpm/l), and were further incubated for 1-10 min at 30°C. Samples were transferred on ice and centrifuged for 5 min at 14,000 ϫ g at 4°C. The membranes were rapidly resuspended in 60 l of labeling buffer (0.1 mM EDTA, 5 mM MgCl 2 , 30 mM NaCl, 50 mM HEPES-KOH, pH 7.5, 1 mM benzamidine, 2 mM glutathione) and immediately irradiated at 265 nm for 10 -15 s at 4°C. The samples were centrifuged as above, and the membranes were solubilized in 160 l of immunoprecipitation buffer (1% Nonidet P40, 1% sodium deoxycholate, 0.5% SDS, 150 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.2 mM PMSF, 10 g/ml aprotinin, and 10 mM Tris-Cl, pH 7.4) for 10 -15 min on ice. Insoluble material was removed by centrifugation as above, and the supernatant was used for immunoprecipitation using an antiserum (5 l) against the common C-terminal decapeptide of G␣s and XL␣s. The samples were incubated with the antibody overnight at 4°C followed by addition of protein A-Sepharose (60 l of a 10% slurry) and further incubation for 2 h at 4°C. The Sepharose beads were pelleted and washed twice with buffer A (1% Nonidet P40, 0.5% SDS, 600 mM NaCl, 50 mM Tris-Cl, pH 7.4) and once with buffer B (300 mM NaCl, 10 mM EDTA, 100 mM Tris-Cl, pH 7.4). Immunoprecipitated material was analyzed by SDS-PAGE and phosphoimaging.
Photoaffinity Labeling Using PC12 Cell Membranes-Membranes were washed once with 50 mM Hepes-NaOH, pH 7.4, and the membrane proteins (50 g) were subjected to photoaffinity labeling with [␣-32 P]AA-GTP as described above in the presence of GTP or ATP as indicated in the figure and with the following modifications. (i) The incubation buffer was 1 mM MgCl 2 and 50 mM NaCl, and GDP was omitted; (ii) no receptor agonist was added; (iii) incubation was for 10 min.

SDS-PAGE
SDS-PAGE (7.5 or 10% gels) and immunoblotting was performed according to standard procedures. Dried gels were either autoradiographed followed by densitometric scanning or exposed to phosphoimager plates, and the intensity of the bands was determined using a Fuji phosphoimager and MacBAS software.
Identification of Mouse XL␣s-The IMAGE cDNA clone 1499201 was identified by BLAST searching with the rat and human XL␣s sequences. It was subsequently obtained from Research Genetics and completely sequenced. The clone encodes the full-length mouse XL␣s protein. The sequence was submitted to GenBank TM (accession number AF116268).

Construction of an XL␣s cDNA for in Vitro Transcription/
Translation-The originally published sequence of XL␣s contains a point mutation in the ␣s-part (1) that leads to a single amino acid exchange (L 3 P) at position 519 (corrected translational start (2); amino acid residue 650 of originally published sequence (1)). This point mutation was not found in XL␣s mRNAs isolated from rat pituitary or PC12 cells as revealed by reverse transcription-polymerase chain reaction analysis (data not shown), suggesting that it reflects an aberration from the physiological XL␣s sequence introduced in the course of construction of the cDNA library from which the original XL␣s cDNA was isolated (1). We therefore constructed an XL␣s cDNA, referred to as XL␣s-wt, in which this point mutation in the ␣s-part of XL␣s was reversed to the physiological sequence. This cDNA was used in the in vitro transcription/translation experiments performed in this study.
In Vitro Translation of XL␣s and G␣s-In vitro translation in the rabbit reticulocyte lysate of a G␣s mRNA, obtained by in vitro transcription of a G␣s cDNA, gave rise to a 46-kDa labeled band (Fig. 1, lane 1). Translation of the in vitro transcribed XL␣s-wt mRNA generated a major band of M r 94,000 and additional bands of lower apparent molecular weight (Fig. 1,  lane 4). Immunoprecipitation of the translation products using an antiserum against the C-terminal decapeptide of G␣s showed that XL␣s (Fig. 1, lane 6) contains the same C-terminal epitope as G␣s (Fig. 1, lane 3). Upon immunoprecipitation using an antiserum against the EPAA repeats in the N-terminal region of the XL domain (1), referred to as anti-XL antibody, only XL␣s mRNA-derived translation products of M r Յ 94,000 (Fig. 1, lane 5), but no G␣s mRNA-derived translation products (Fig. 1, lane 2), were obtained. This showed that (i) the anti-XL antibody was indeed specific for the XL domain of XL␣s, and (ii) the major XL␣s mRNA-derived translation products of M r Ͻ 94,000 (Fig. 1, lanes 4 and 5) were truncated in the ␣s domain. In vitro translated G␣s and XL␣s, as shown in Fig.  1, lanes 1 and 4, were used in the subsequent experiments.
Trypsin Resistance of the ␣s Domain of XL␣s in the GTPbound State-The activation of G␣ subunits, i.e. the replacement of GDP by GTP, is associated with a conformational change that is reflected in their differential sensitivity to degradation by trypsin (3). As shown in Fig. 2, the presence of GTP␥S affected the tryptic digestion of in vitro translated XL␣s in the same way as it was described previously for G␣s (3). In the absence of GTP␥S, XL␣s, like G␣s, was fully degraded by increasing concentrations of trypsin. In contrast, for both G␣s and XL␣s in the presence of GTP␥S, 37-35-kDa fragments were protected from tryptic digestion. This strongly suggests that (i) XL␣s binds GTP␥S and (ii) upon GTP␥S binding, the ␣s domain undergoes the same conformational change as G␣s.
Formation of an XL␣s-␤␥ Heterotrimer-In XL␣s, the exon 1-encoded portion of G␣s is replaced by the XL domain (1). The exon 1-encoded portion of G␣s is involved in the interaction of G␣s with the ␤␥ complex (5, 11). Interestingly, the C-terminal region of the XL domain shows a high degree of homology to the exon 1-encoded portion of G␣s and other types of G␣ subunits (1) (Fig. 3, A and B). In particular, the residues that are known to directly contact the ␤␥ dimer (11,12) are conserved not only between rat, mouse, and human XL␣s (Fig. 3B) but also between XL␣s and the various types of G␣ subunits (1). It is therefore possible that XL␣s, like G␣s, binds to the ␤␥ complex. To investigate this issue, we used (i) ADP-ribosylation by chol-era toxin to detect even weak or transient XL␣s-␤␥ interactions and (ii) sucrose density gradient centrifugation to search for stable heterotrimer formation.
Effect of ␤␥ Subunits on the ADP-ribosylation of XL␣s by Cholera Toxin-The ability of activated cholera toxin to catalyze the ADP-ribosylation of purified (i.e. monomeric), native G␣s (13) as well as of in vitro translated G␣s (3) is very poor. However, in either case, the addition of ␤␥ subunits before the addition of activated cholera toxin greatly increases the extent of ADP-ribosylation (3,13). ADP-ribosylation by cholera toxin is an irreversible modification and is therefore a very sensitive method for detecting even weak or transient interactions of in vitro translated G␣s and XL␣s with ␤␥ subunits. In the absence of ␤␥ subunits, in vitro translated XL␣s was indeed found to be a poor substrate for cholera toxin catalyzed ADP-ribosylation (Fig. 4, lane 4), whereas the addition of exogenous ␤␥ subunits resulted in a Ͼ2.2-fold increase in the labeling of both the in vitro translated XL␣s (Fig. 4, lane 5) as well as the endogenous G␣s present in the reticulocyte lysate (Fig. 4, lanes 2 and 5). The labeling of XL␣s and G␣s in the absence of added ␤␥ subunits (Fig. 4, lanes 1 and 4) is most likely due to the presence of some endogenous ␤␥ in the reticulocyte lysate (14).
Effect of ␤␥ Subunits on the Sedimentation Behavior of XL␣s-To look for stable heterotrimer formation, we examined the effects of purified ␤␥ subunits on the sedimentation behavior of in vitro translated XL␣s and, for comparison, G␣s, using sucrose density gradients. In the absence of ␤␥ subunits, XL␣s (Fig. 5B, open circles) and G␣s (Fig. 5A, open circles) sedimented at a rate corresponding to a calculated molecular mass of 78 and 46 kDa, respectively, and hence a monomeric state, as revealed by comparison to the 68-kDa hemoglobin tetramer (Fig. 5, bars). In the presence of ␤␥ subunits, XL␣s (Fig. 5B, filled circles) and confirming previous results (5), G␣s (Fig. 5A, filled circles), sedimented at a faster rate. However, in the presence of ␤␥ subunits, XL␣s (Fig. 5B, filled circles) sedimented at a slower rate than G␣s (Fig. 5A, filled circles), although the total molecular mass of an XL␣s-␤␥ heterotrimer is greater than that of the G␣s-␤␥ heterotrimer. This suggests that XL␣s, upon contact with the ␤␥ dimer, undergoes a conformational change, which alters its sedimentation behavior.
Activation of Adenylyl Cyclase by a GTPase-deficient Mutant of XL␣s-To determine whether XL␣s is capable of activating adenylyl cyclase, we constructed a GTPase-deficient mutant of XL␣s, XL␣sQ548L. This mutation corresponds to the Q227L mutation in G␣s, which is analogous to the Q61L mutant of p21 ras . In G␣s, this mutation leads to a 100-fold reduction in the rate constant of GTP hydrolysis (15). Hence, XL␣sQ548L should be constitutively activated.
Immunoblotting of PC12 cell membranes using the antibody against the common C-terminal decapeptide of G␣s and XL␣s (Fig. 6, bottom panel) indicated that transient transfection with the cDNA for XL␣s or XL␣s-Q548L substantially increased the amount of XL␣s above the endogenous level. Membranes of transfected and untransfected PC12 cells were then analyzed for adenylyl cyclase activity (Fig. 6, top panel). Cells transfected with the activated form of XL␣s, XL␣s-Q548L, showed a massive increase in adenylyl cyclase activity as compared with wild type or mock-transfected cells. Transfection of XL␣s resulted in only a small increase in adenylyl cyclase activity, presumably because XL␣s was predominantly in the GDPbound state and therefore inactive toward adenylyl cyclase. The addition of forskolin to the membranes from wild type, mock-transfected, and XL␣s-transfected PC12 cells increased adenylyl cyclase activity to the level observed with membranes from XL␣s-Q548L-transfected cells (data not shown), showing that adenylyl cyclase in the former membranes could be acti-  1 and 2) or containing (lanes 3-5) in vitro translated XL␣s was incubated for 60 min at 30°C with 32 P-NAD ϩ in the absence (Ϫ) or presence (ϩ) of purified ␤ 1 ␥ 2 subunits and cholera toxin (Ctx) as indicated, followed by immunoprecipitation with the antiserum against the C-terminal decapeptide of G␣s and XL␣s. Immune complexes were analyzed by SDS-PAGE and autoradiography. The results shown are representative of three independent experiments. vated. When HeLa (rather than PC12) cells were transiently transfected with XL␣s-Q548L, they also showed an increase (4-fold) in adenylyl cyclase activity as compared with mocktransfected cells (data not shown).
Receptor Activation of G␣s, but Not XL␣s, in Reconstituted S49cyc Ϫ Membranes-S49cyc Ϫ cells are deficient in G␣s (16 -18) as well as XL␣s, as revealed by immunoblotting (data not shown), but still express the ␤2-adrenergic receptor, ␤␥ subunits, and the adenylyl cyclase. Given that XL␣s can activate adenylyl cyclase (Fig. 6), we used S49cyc Ϫ cells to study the signal transduction properties of XL␣s. As reported previously (5,8), adenylyl cyclase activity of S49cyc Ϫ membranes could be stimulated upon the addition of isoproterenol, a ␤2-adrenergic receptor agonist, when the membranes had been reconstituted with in vitro translated G␣s (Fig. 7A). Compared with the addition of GTP alone, adenylyl cyclase activation upon receptor stimulation was increased to about half of the maximal value obtained in the presence of GTP␥S (Fig. 7A).
Because G␣s and XL␣s contain the same C-terminal domain and may therefore couple to the same type of receptor (19 -21), we investigated whether XL␣s, like G␣s, was able to restore receptor stimulation of adenylyl cyclase activation in reconstituted S49cyc Ϫ membranes. In contrast to G␣s (Fig. 7A), XL␣s mediated only a small, barely significant increase in adenylyl cyclase activation upon receptor stimulation, as compared with the addition of GTP or GTP␥S alone (Fig. 7B). We therefore conclude that, in this in vitro system, XL␣s couples to the ␤2-adrenergic receptor much less efficiently than G␣s, if at all. Consistent with these in vitro findings, we observed, in comparison with untransfected PC12 cells, an increased adenylyl cyclase activity upon the addition of 100 M CGS 21680 (adenosine 2A receptor agonist) in membranes of G␣s-transfected, but not XL␣s-transfected, PC12 cells (data not shown).
Receptor Activation of G␣s, but not XL␣s, in Pituitary Membranes-Given that XL␣s couples only weakly to the ␤2 adrenergic receptor in reconstituted S49cyc Ϫ membranes, we investigated whether or not XL␣s is capable of coupling to a G protein-coupled receptor in vivo. For this purpose, we used photoaffinity labeling of G protein ␣ subunits with AA-GTP (10). When combined with immunoprecipitation using antibodies specific for a given G protein ␣ subunit, such photoaffinity labeling is a powerful tool to identify the specific G protein ␣ subunit activated by a given receptor.
First, we investigated whether XL␣s binds AA-GTP with the same affinity as G␣s. PC12 cell membranes were incubated in the presence of AA-GTP with increasing concentrations of either unlabeled GTP or ATP. As shown in Fig. 8A,  AA-GTP to XL␣s could be competed with increasing concentrations of GTP but not ATP. The comparison of the competition profiles obtained for XL␣s and G␣s shows that XL␣s binds to AA-GTP with virtually the same affinity as G␣s.
Second, we determined whether AA-GTP binding to XL␣s could be enhanced by the activation of known G␣s-coupled receptors. For this purpose, we used rat pituitary membranes because the expression level of XL␣s was highest in this tissue (32). Fig. 7B shows that the receptor for pituitary adenylyl cyclase-activating polypeptide (PACAP) was capable of activating G␣s, as indicated by an increased incorporation of AA-GTP in the presence of PACAP but not XL␣s.
Other G␣s-coupled receptors in the pituitary, i.e. that for vasoactive intestinal polypeptide (VIP, used at 1-10 M) and corticotropin releasing factor (CRF, used at 10 M) as well as the adenosine 2A receptor of PC12 cells (22,23) (stimulated by 10 M CGS 21680), were also found to activate G␣s but not XL␣s (data not shown). Changing the experimental conditions, e.g. by the addition of various concentrations of GDP to suppress the basal rate of guanine nucleotide exchange, or using different labeling times also did not reveal any significant receptor stimulation of guanine nucleotide exchange on XL␣s (data not shown). DISCUSSION Our study shows that XL␣s shares many, but not all, functional properties of G␣s. XL␣s (i) forms a heterotrimer with ␤␥ subunits, (ii) binds GTP and undergoes a conformational change upon GTP binding, and (iii) activates, when in the GTP state, adenylyl cyclase. However, XL␣s does not appear to be activated by known G␣s-coupled receptors.
Binding to the ␤␥ Dimer-We used two methods to demonstrate the ability of XL␣s to interact with ␤␥ subunits: (i) ADP-ribosylation by cholera toxin, which in the case of G␣s is greatly promoted by its heterotrimeric state (3,13,24), and (ii) sucrose density gradient centrifugation. The addition of ␤␥ subunits to in vitro translated XL␣s increased its ADP-ribosylation as well as its sedimentation rate in sucrose density gradients, indicating that XL␣s forms heterotrimers with ␤␥ subunits. Remarkably, however, comparison of the sedimentation behavior of G␣s and XL␣s in the presence of ␤␥ dimers revealed that XL␣s sedimented more slowly than G␣s, although the molecular mass of an XL␣s-␤␥ heterotrimer is greater than that of a G␣s-␤␥ heterotrimer. The observations that (i) heterotrimeric rather than monomeric G␣s (3,13,24) and XL␣s (Fig. 4) is a substrate for cholera toxin-catalyzed ADP-ribosylation and (ii) XL␣s, like G␣s, undergoes ADP-ribosylation by cholera toxin in vivo (1) strongly suggest that XL␣s exists in the heterotrimeric state in vivo. It is therefore likely that the sedimentation of XL␣s observed in the presence of ␤␥ subunits reflected that of heterotrimeric XL␣s rather than that of a monomeric XL␣s that had adopted a different conformation upon transient contact with the ␤␥ dimer. This in turn suggests that the heterotrimerization of XL␣s with the ␤␥ dimer is associated with a stable conformational change of XL␣s toward a more rod-like state, resulting in a slower sedimentation of the XL␣s-␤␥ heterotrimer than the G␣s-␤␥ heterotrimer. The proline-rich region of the XL domain of XL␣s (1) (Fig. 3, A and C) could serve as a hinge for this conformational change. It is worth noting that this region, like the C-terminal region of the XL domain implicated in ␤␥ binding (1) (Fig. 3, A and B) is more highly conserved in XL␣s of various species (Fig. 3C) than other regions of the XL domain (for a comparison of human and rat XL␣s, see Hayward et al. (25)).
The ability of XL␣s to form a heterotrimeric complex with ␤␥ subunits also has implications for the observations reported in the preceding paper (32) that immunoreactive and ADP-ribo-sylatable XL␣s show a distinct distribution upon subcellular fractionation, whereas this is not the case for G␣s. Specifically, the subpopulation of XL␣s molecules that were poorly, if at all, ADP-ribosylated by cholera toxin were preferentially recovered in fractions containing plasma membrane, whereas the subpopulation that was well ADP-ribosylated was preferentially recovered in fractions containing Golgi membranes and certain subdomains of the plasma membrane (see preceding paper (32)). This raises the possibility of an interplay between ␤␥ binding to XL␣s and its subcellular localization, which does not take place for G␣s.
GTP Binding and Functional Units-Photoaffinity labeling showed that XL␣s binds GTP. The ␣s domain of XL␣s is likely folded in the same way as G␣s and, upon GTP binding, undergoes the same conformational change as G␣s, since the pattern FIG. 8. Differential effect of PACAP receptor stimulation on GTP binding to XL␣s and G␣s. A, PC12 cell membranes were incubated for 10 min with [␣-32 P]GTP-azidoanilide in the presence of the indicated concentrations of unlabeled GTP or ATP, followed by photocross-linking. XL␣s and G␣s were immunoprecipitated from the solubilized membranes using an antiserum against the common C-terminal decapeptide, and immunecomplexes were analyzed by SDS-PAGE followed by autoradiography. Top, XL␣s region of the autoradiogram. Bottom, quantitation of the 32 P-labeled XL␣s (filled circles) and G␣s (open circles) by densitometric scanning; a.u., arbitrary units. To facilitate comparison with XL␣s, the G␣s values (sum of two G␣s bands) were divided by 2.5 (GTP) and 2.2 (ATP). B, rat pituitary membranes were incubated for the indicated times with [␣-32 P]GTP-azidoanilide in the presence of 10 M GDP in the absence (Ϫ) or presence (ϩ) of 1 M PACAP, followed by photocross-linking. XL␣s and G␣s were immunoprecipitated from the solubilized membranes using an antiserum against the common C-terminal decapeptide, and immune complexes were analyzed by SDS-PAGE and phosphoimaging (top). Bottom, quantitation of the 32 P-labeled XL␣s and G␣s. For each period of incubation, the value obtained in the absence (Ϫ) of PACAP was arbitrarily set to 100, and the value obtained in the presence (ϩ) of PACAP was expressed relative to this. The results shown are representative of five independent experiments. of the trypsin-resistant fragments was identical for G␣s and XL␣s. This implies that the XL domain does not significantly affect the folding of the ␣s domain of XL␣s and its conformational change upon GTP binding.
Together, our data suggest that XL␣s is composed of two independent units that are connected by the proline-rich region of the XL domain (Fig. 3A), a putative hinge (see above). Although encoded by the XL exon, the cysteine-rich region of the XL domain (Fig. 3A) (1), which is palmitoylated and hence contributes to membrane attachment of XL␣s (see Ugur and Jones (26) and the preceding paper (32)) and the C-terminal region of the XL domain, which is implicated in ␤␥ binding (Fig.  3A), are suggested to belong, together with the ␣s domain, to one unit that exerts all G␣s-type functions of XL␣s. The other unit, whose function remains to be elucidated, comprises the alanine-rich, repetitive region of the XL domain (Fig. 3A) (1), which does not show a high sequence conservation between rat, mouse, and human XL␣s (data not shown; for a comparison of human and rat XL␣s, see Hayward et al. (25)).
Activation of Adenylyl Cyclase-Overexpression of a constitutively active mutant of XL␣s in PC12 cells led to a massive increase in cAMP production, showing that XL␣s-GTP, via its G␣s-type unit, is capable of activating adenylyl cyclase. Hence, in neuroendocrine cells, two distinct G protein ␣-subunits, G␣s and XL␣s, converge onto the same effector system, adenylyl cyclase.
Receptor Coupling-The C-terminal domain of a G␣ subunit is not only critical for the interaction with a receptor (27)(28)(29) but also a determinant of the specificity of G protein-receptor interaction (19 -21). Given that (i) the C-terminal domain of XL␣s is identical to G␣s and (ii) XL␣s is able to form a heterotrimeric complex with ␤␥ subunits, which in the case of typical G␣ subunits is a prerequisite for their interaction with a receptor, one might have expected XL␣s to become activated by G␣s-coupled receptors. Remarkably, however, using various experimental approaches including reconstitution of S49cyc Ϫ membranes, AA-GTP photoaffinity labeling, and transfection of cells, all of which allowed the detection of receptor-mediated activation of G␣s, we have been unable to obtain conclusive evidence for receptor-mediated activation of XL␣s.
It is possible that XL␣s is not subject to activation by any receptor. However, the apparent lack of receptor-mediated activation of XL␣s may also reflect a novel, as yet not understood, mode of activation (rather than the wrong choice of receptors examined in this study). If so, it is tempting to speculate that the presence of the alanine-rich, repetitive region of the XL domain, which is located N-terminal to the G␣s-type unit, provides another level of regulation, for example by sterically hindering in a reversible manner the coupling of the ␣s domain of XL␣s to a receptor.