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Endocrinology Vol. 139, No. 2 626-633
Copyright © 1998 by The Endocrine Society

ARTICLES

Altered G Protein Activity in a Desensitization-Resistant Mutant of the Y1 Adrenocortical Tumor Cell Line1

Concettina M. Colantonio2, Wai-King Kwan, Waldemar Czerwinski, Jane Mitchell and Bernard P. Schimmer

Banting and Best Department of Medical Research (C.M.C., W.-K.K., W.C., B.P.S.) and the Department of Pharmacology (C.M.C., J.M., B.P.S.), University of Toronto, Toronto, Ontario, Canada M5G 1L6

Address all correspondence and requests for reprints to: Dr. Bernard P. Schimmer, Banting and Best Department of Medical Research, University of Toronto, 112 College Street, Toronto, Ontario, Canada M5G 1L6. E-mail: bernard.schimmer{at}utoronto.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mutant isolates [designated desensitization resistant (DR)] from the Y1 mouse adrenocortical tumor cell line resist agonist-induced desensitization of adenylyl cyclase by preventing the uncoupling of receptors from their guanyl nucleotide-binding regulatory G proteins. In this study, we tested the hypothesis that an underlying G protein defect is associated with the DR phenotype. We found that the G protein reagent guanyl-5'-yl imidodiphosphate [Gpp(NH)p] shifted ß2-adrenergic receptors from a high affinity state to a low affinity state 4-fold more effectively in mutant DR cells than in parent Y1 cells. In the DR mutant, Gpp(NH)p was able to shift receptors to a low affinity state in the absence of NaCl, whereas the effect of Gpp(NH)p in parent Y1 cells was dependent upon the presence of NaCl. Moreover, these differences in sensitivity to Gpp(NH)p and NaCl were transferred to Gs{alpha}-deficient S49(CYC-) lymphoma cell membranes in G protein reconstitution assays. These observations suggested that the DR mutation was associated with altered activity of the stimulatory G protein, Gs. Cloning and sequence analysis demonstrated that Gs{alpha} transcripts in the DR mutant were normal, suggesting that another factor involved in guanyl nucleotide exchange is responsible for the altered G protein activity in DR mutant cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CELLS chronically stimulated by hormones or neurotransmitters eventually undergo an adaptive process that renders them insensitive to further stimulation. This phenomenon, termed agonist-induced desensitization, has been studied extensively in cells expressing G protein-coupled receptors, in which desensitization leads to a decreased ability of specific agonists to activate effectors such as adenylyl cyclase (for review, see Ref.1). Considerable emphasis has been placed on receptor modifications such as phosphorylation, depalmitoylation, and internalization in the desensitization process and on the role of arrestins that uncouple phosphorylated receptors from various G proteins (see Ref. 2 for review). There is growing evidence to suggest that G proteins also may play important roles in the desensitization process. G protein modifications such as acylation/deacylation (3), phosphorylation (4), redistribution, and down-regulation (5, 6, 7) accompany agonist-induced desensitization, and regulators of G protein signaling have been identified that promote G protein guanosine triphosphatase activity and promote agonist-induced desensitization (8, 9).

As part of our efforts to understand the mechanisms that contribute to agonist-induced desensitization of adenylyl cyclase, we described a family of mutants [desensitization resistant (DR)] derived from the Y1 mouse adrenocortical tumor cell line that resisted desensitization induced by the peptide hormone ACTH (10). We have characterized one of these mutants extensively in an attempt to identify the underlying cause of the DR mutation and potentially to describe additional genes that regulate desensitization. Previously, we showed that the DR mutant resisted homologous desensitization by ß-adrenergic agonists when transfected with a gene encoding the mouse ß2-adrenergic receptor (ß2AR) (11) and resisted heterologous desensitization via dopaminergic agonists when transfected with a gene encoding the human dopamine D1 receptor (12). On this basis, we concluded that the DR mutation was not a receptor-associated defect, but instead affected a part of the desensitization pathway that was shared by the ACTH receptor, the ß2AR, and the dopamine D1 receptor and was common to homologous and heterologous desensitization pathways. We also showed that receptor sequestration in parental desensitization-sensitive (DS) and mutant DR cells did not differ (11), indicating that changes in receptor internalization and down-regulation were not responsible for the differences between DS and DR clones. Characterization of the ligand-binding properties of ß2ARs in transfected DR cells indicated that the DR mutation protected cells from desensitization by preventing agonist-induced uncoupling of receptors from their signal-transducing G proteins and raised the possibility that the DR phenotype resulted from an underlying G protein defect (13).

In the present study, we have assessed G protein activity in DS and DR cells by comparing the abilities of the G protein reagents guanyl-5'-yl imidodiphosphate [Gpp(NH)p] and NaCl to modulate receptor-G protein interactions. We find that receptor-G protein interactions in DS and DR cells exhibit different sensitivities to the uncoupling effects of these reagents. Furthermore, these differences are transferred to Gs{alpha}-deficient S49(CYC-) membranes in G protein reconstitution assays. Based on complementary DNA (cDNA) cloning and sequence analysis, we also show that transcripts encoding the long form of Gs{alpha} (14), the principal isoform expressed in DS and DR cells, are identical, indicating that the desensitization-resistant phenotype of DR mutant Y1 adrenocortical tumor cells does not result from a Gs{alpha} mutation, but, rather, from an underlying mutation affecting G protein function.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
[125I]Iodocyanopindolol, ECL chemiluminescent detection system, and peroxidase-labeled antirabbit secondary antibody were obtained from Amersham Canada (Oakville, Canada). Schleicher and Schuell nitrocellulose membranes were obtained from Xymotech Biosystems (Toronto, Canada). Isoproterenol and Gpp(NH)p were obtained from Sigma Canada (Missisauga, Canada), Lubrol Dx was purchased from ICN Canada (Montreal, Québec, Canada), and DMEM with high glucose was purchased from the University of Toronto Media Center (Toronto, Canada). Other culture media, sera, oligonucleotides, the 5'-RACE (rapid amplification of 5'-ends) system, and Superscript II reverse transcriptase were obtained from Canadian Life Technologies (Burlington, Canada). Sequenase 2.0 was purchased from U.S. Biochemical Corp. (Cleveland, OH). S49 and S49CYC- cells were received from the University of California Cell Culture Facility (San Francisco, CA).

Cells and cell culture
Parental Y1 adrenocortical tumor cells (DS), a representative DR mutant (10), and two independent ß2AR transformants of each of the DS and DR cell lines (11) were cultured as monolayers at 36.5 C under a humidified atmosphere of 95% air-5% CO2 in nutrient mixture F-10 supplemented with 15% heat-inactivated horse serum, 2.5% heat-inactivated FBS, and antibiotics. S49 mouse lymphoma cells and the S49CYC- mutant (15) were grown as suspension cultures at 36.5 C in DMEM supplemented with high glucose (4.5 g/liter), 10% heat-inactivated horse serum, and antibiotics.

Preparation of cell membranes
Y1 cells and derivative clones were grown as monolayers on 15-mm tissue culture dishes to saturation density and then incubated in {alpha}MEM supplemented with 1% heat-inactivated FBS for 18–24 h. S49 and S49CYC- lymphoma cells were grown as suspension cultures in 75-mm tissue culture bottles to saturation density and harvested by centrifugation at 400 x g for 5 min at 4 C. Cells were washed three times with buffer (1 mM MgCl2, 250 mM sucrose, and 20 mM Tris-HCl, pH 7.7), and homogenized in ice-cold binding buffer (5 mM MgCl2, 1.5 mM CaCl2, 5 mM EDTA, 5 mM KCl, and 50 mM Tris-HCl, pH 7.4) at 0 C using a Dounce homogenizer (Kontes Co., Vineland, NJ) with a tight-fitting pestle. The homogenates were centrifuged at 800 x g for 5 min at 4 C to remove nuclei, and intact cells and cell membranes then were pelleted by centrifugation at 40,000 x g for 15 min at 4 C. The membrane fraction was resuspended in binding buffer and stored frozen at -70 C before use.

Ligand binding assays
Ligand binding assays were carried out on membrane fractions using [125I]iodocyanopindolol as the labeled ligand and the ß-adrenergic agonist, isoproterenol, as the unlabeled competitor. Approximately 100 µg membrane protein were added to tubes containing 40 pM [125I]iodocyanopindolol (~70,000 dpm; 2,000 Ci/mmol), varying concentrations of Gpp(NH)p (0–200 µM) with or without 120 mM NaCl, and varying concentrations of isoproterenol (0–10-4 M) in a final volume of 0.5 ml and incubated at 37° C for 30 min. Incubations were terminated by filtration through glass fiber receptor-binding filter mats (Skatron, Sterling, VA). Filters were washed with 5 ml binding buffer at room temperature, and the amount of radioactive ligand bound was measured by {gamma}-scintillation spectrometry. Binding parameters were determined using the program Ligand (16).

Reconstitution of G protein activity in S49CYC- membranes
Cell membranes prepared as detailed above were centrifuged at 40,000 x g for 5 min at 4 C and resuspended in extraction solution (10 mM MgCl2, 0.1 mM EDTA, 1 mM dithiothreitol, 0.2% Lubrol Dx, and 10 mM Tris-HCl, pH 7.5) at protein concentrations ranging from 0.5–4.0 mg/ml. Mixtures were placed on ice for 30 min to extract G proteins and then were centrifuged at 40,000 x g for 5 min at 4 C. The solubilized G protein fractions were used to reconstitute S49CYC- membranes essentially as detailed previously (17). G protein extracts were added to S49CYC- membranes (10 mg/ml in 10 mM Tris-HCl, pH 7.5) at a ratio of 1:2 (vol/vol). The mixture was gently triturated in a 1-ml pipette and centrifuged at 40,000 x g for 5 min at 4 C. The resulting pellet was resuspended in binding buffer to a final protein concentration of 1 mg/ml and represented the G protein-reconstituted membrane fraction.

Western blot analysis of Gs{alpha}
Membrane fractions were solubilized in 100 mM dithiothreitol, 2% SDS, and 50 mM Tris-HCl, pH 6.8, and electrophoresed on 10% polyacrylamide gels in 25 mM Tris-250 mM glycine buffer containing 0.1% SDS. Resolved proteins were electroblotted onto nitrocellulose membranes and subjected to Western blot analysis for Gs{alpha} using a chemiluminescent detection system. Briefly, the blots were blocked with Tris-buffered saline (TBS)-Tween (137 mM NaCl, 0.05% Tween-20, and 20 mM Tris-HCl, pH 7.6) containing 5% skim milk powder and incubated for 2 h at room temperature with rabbit A572 anti-Gs{alpha} serum (18) diluted 1:500 in blocking buffer. The blots were washed with TBS-Tween and then incubated for 1 h at room temperature with horseradish peroxidase-labeled secondary antirabbit antibodies diluted 1:10,000 in blocking buffer. Membrane filters were washed in TBS-Tween, and antibody interactions with target proteins were visualized by chemiluminescence after a 20-min exposure to Reflection autoradiograph film (DuPont Canada, Missisauga, Canada).

Isolation and sequence analysis of Gs{alpha} cDNA
Total RNA was extracted from DS and DR cells with guanidine thiocyanate and pelleted by centrifugation through CsCl (19). Gs{alpha} cDNA was synthesized from total RNA using a primer (bases 1294–1275 relative to the initiator ATG) complementary to the 3'-untranslated region of {alpha}s-3 (20) and Superscript II reverse transcriptase. Gs{alpha} cDNA was then amplified in two overlapping fragments by PCR using a kit containing AmpliTaq DNA polymerase [Perkin-Elmer (Canada), Rexdale, Canada]. A cDNA fragment corresponding to the 3'-half of Gs{alpha} was amplified with a forward primer from 603–622 and a reverse primer from 1294–1275; a 5'-Gs{alpha} cDNA fragment was amplified with a forward primer from 66–85 and a reverse primer from 814–795. PCR was carried out for 30–35 cycles with a hot start (21); the timing for each cycle consisted of 1-min incubations at 94, 60, and 72 C. At the end of the reaction, samples were incubated for 10 min at 72 C to ensure that cDNA synthesis proceeded to completion. The remaining 5'-sequence of Gs{alpha} was obtained by 5'-RACE (22) using a gene-specific reverse primer corresponding to the Gs{alpha} cDNA sequence from 399–380.

cDNA fragments were subcloned into the pT7Blue-T vector (Novagen, Madison, WI), and double-stranded cDNA was prepared from at least nine subclones of each fragment. The cDNAs were sequenced by the dideoxynucleotide chain termination method (23) using Sequenase 2.0 and sequencing primers derived either from vector sequences flanking the cDNA inserts or from internal Gs{alpha} sequences.

Statistical analysis
Unless otherwise indicated, statistical significance of differences was determined by one- and two-way ANOVA with independent samples.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Receptor/G protein coupling in parent DS and mutant DR cells
Receptor/G protein coupling was assessed in membranes of ß2AR-transformed DS (DSß2+) and DR (DRß2+) cells by estimating the proportion of agonist-binding sites in the high affinity state. Agonist displacement curves generated with DRß2+ and DSß2+ membranes each described two populations of receptors, one in a high affinity state and one in a low affinity state (Table 1Go). The proportion of receptors in the high affinity state was significantly greater for DRß2+ membranes (59.8 ± 6.6%) than for DSß2+ membranes (35.9 ± 8.9%; P < 0.01. In both DSß2+ and DRß2+ membranes, all of the receptors were shifted to a low affinity state after treatment with 200 µM Gpp(NH)p, indicating that high concentrations of the guanyl nucleotide effectively uncoupled ß2ARs from their G proteins (Fig. 1Go and Table 1Go). The ß2ARs in the DR transformants, however, seemed to uncouple from their corresponding G proteins at much lower concentrations of Gpp(NH)p than did receptors in the DS transformants. Agonist displacement curves generated using DRß2+ membranes were shifted extensively to the right in the presence of 25 µM Gpp(NH)p, whereas displacement curves generated using DSß2+ membranes were not affected by this low concentration of guanyl nucleotide (Fig. 1Go). The results presented in Fig. 2Go describe in more detail the concentration dependence of Gpp(NH)p on receptor/G protein coupling in membranes from two DSß2+ transformants and two DRß2+ transformants, thus emphasizing the difference in sensitivity of DSß2+ and DRß2+ cells to the uncoupling effects of Gpp(NH)p. As shown, 25 µM Gpp(NH)p was sufficient to shift all ß2ARs in DRß2+ membranes from high affinity to low affinity, whereas a 4-fold greater concentration of Gpp(NH)p was required to convert the ß2ARs of DSß2+ membranes to the low affinity state.


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  		Table 1. Binding parameters for ß2-ARs in DR and DS transformants

 


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  		Figure 1. Agonist displacement curves using membranes from ß2AR-transformed DS and DR cells. ß2ARs were analyzed in membrane preparations from a representative DS transformant (A) and a representative DR transformant (B) using a competition binding assay as detailed in Materials and Methods. The antagonist [125I]iodocyanopindolol (ICYP) was the labeled ligand, and the agonist isoproterenol was the unlabeled competitor. Binding analyses were performed on membranes with 0 µM Gpp(NH)p ({square}), 25 µM Gpp(NH)p plus 120 mM NaCl (•), or 200 µM Gpp(NH)p plus 120 mM NaCl ({triangleup}) as indicated. Data are expressed as a percentage of specifically bound ICYP vs. the concentration of competing isoproterenol. The results shown are representative of four independent experiments.

 


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  		Figure 2. Sensitivities of ß2ARs in DS and DR transformants to Gpp(NH)p. The ligand-binding characteristics of ß2ARs in membrane preparations from two DS (open and closed circles) and two DR (open and closed squares) transformants were analyzed in the presence of varying concentrations of Gpp(NH)p plus 120 mM NaCl as described in Fig. 1Go. The percentage of receptors in the high affinity state in the absence of Gpp(NH)p and NaCl (~36% for DS and 60% for DR transformants) was normalized to 1.0 for each clone. Data are presented as the proportion of high affinity binding sites ± SEM.

 
Effects of NaCl on receptor/G protein coupling
In addition to Gpp(NH)p, NaCl reportedly is required to maximally shift ß2ARs from a high affinity state to a low affinity state (24, 25). For this reason, NaCl was included together with Gpp(NH)p in the binding experiments described in Figs. 1Go and 2Go. To assess the effects of NaCl on the ligand-binding properties of ß2ARs in DSß2+ and DRß2+ membranes, agonist displacement curves were generated in the presence of 200 µM Gpp(NH)p with or without 120 mM NaCl. As shown in Fig. 3Go, agonist competition curves generated using DSß2+ membranes were shifted to the right only when Gpp(NH)p and NaCl were both present in the binding assay. Computer-assisted analysis of the agonist displacement curves indicated that the rightward shift seen with Gpp(NH)p and NaCl reflected the complete conversion of high affinity receptor-binding sites to low affinity (P < 0.05), whereas Gpp(NH)p alone was unable to decrease the apparent affinity of the ß2ARs for agonist in DSß2+ membranes (P > 0.05). In contrast, agonist displacement curves generated with DRß2+ membranes were shifted to the right with Gpp(NH)p alone, reflecting a complete conversion of receptors to the low affinity state. The addition of NaCl to the binding assay had no additional effect on the apparent affinity of the ß2ARs for agonist. Similar results were obtained with a second independent set of DSß2+ and DRß2+ transformants (data not shown). Although the precise mechanisms by which NaCl regulates receptor affinity are unknown, direct actions on ß2AR (26), G protein guanosine triphosphatase activity (27), and GTP dissociation (28) have been observed.



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  		Figure 3. Effects of NaCl on agonist binding properties of DS and DR membranes. ß2ARs in membranes from a DS (A) and a DR (B) transformant were analyzed in competition binding assay as described in Fig. 1Go. Binding analyses were performed in the absence of Gpp(NH)p ({square}), with 200 µM Gpp(NH)p in the absence of NaCl ({blacktriangleup}), and with 200 µM Gpp(NH)p plus 120 mM NaCl ({triangleup}). Results are from a single experiment and are representative of data obtained with two independent DS and DR transformants assayed at least in duplicate.

 
Reconstitution of DS and DR phenotypes in S49CYC- lymphoma membranes
The different sensitivities of DSß2+ and DRß2+ membranes to Gpp(NH)p and NaCl were consistent with the hypothesis that the DR phenotype was associated with altered G protein function. To further test this correlation and to examine the contribution of Gs{alpha} to the DR phenotype, G proteins were extracted from the membranes of untransformed (i.e. ß2AR-) DS and DR cells and assayed for activity following reconstitution into Gs{alpha}-deficient S49CYC- lymphoma membranes. G protein extracts prepared from DS and DR cells successfully restored isoproterenol-responsive and NaF-responsive adenylyl cyclase activity to S49CYC- membranes. The levels of basal and isoproterenol-stimulated adenylyl cyclase activities obtained in reconstituted membranes using G protein extracts from DR cells were consistently higher than the levels obtained using extracts from DS cells; differences in NaF-stimulated adenylyl cyclase activity using DS and DR extracts did not reach statistical significance (Table 2Go).


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  		Table 2. Adenylyl cyclase activity in reconstituted S49CYC- membranes

 
As shown in Fig. 4Go, G protein extracts from DR membranes converted the endogenous ß2ARs associated with the S49CYC- membranes to a high affinity state much more readily than did extracts from DS membranes (P < 0.01). G proteins extracted from DR membranes at concentrations of 1 mg membrane protein/ml shifted 49 ± 6% of the ß2ARs to a high affinity state and produced a near-maximum effect. In contrast, G protein extracts from DS membranes at concentrations lower than 3 mg/ml failed to convert ß2ARs in S49CYC- membranes to high affinity ligand binding sites. Furthermore, the proportion of ß2ARs in the high affinity state were greater in S49CYC- membranes reconstituted with DR extracts than in membranes reconstituted with DS extracts (Fig. 4Go). As determined from Western blot analyses (Fig. 5Go), the amounts of Gs{alpha} extracted from DS and DR membranes did not differ, nor did the amounts of Gs{alpha} recovered from S49CYC- membranes differ after reconstitution with extracts from DS or DR membranes. Taken together, these latter results indicate that the different abilitites of G protein extracts to convert ß2ARs to a high affinity state and to regulate adenylyl cyclase activity reflected differences in Gs{alpha} activity rather than differences in Gs{alpha} extraction from the adrenal cell membranes or uptake by S49CYC- membranes.



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  		Figure 4. Reconstitution of high affinity binding sites in S49CYC- membranes with G protein extracts from DS and DR cells. S49CYC- membranes were reconstituted with varying concentrations of G protein extract prepared from the untransformed DS ({circ}) or DR ({square}) cells and assayed for ß2ARs in competition binding assays as described in Fig. 2Go. Results are expressed as the percentage of ß2ARs in the high affinity state and are presented as the mean ±SD of three or more determinations.

 


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  		Figure 5. G Protein levels in cell extracts and reconstituted S49CYC- membranes. A, Membranes from parent DS and DR cells were extracted with nonionic detergent at different protein concentrations and analyzed for Gs{alpha} content as described in Materials and Methods. Gs{alpha} purified from rabbit liver served as a positive control. B, G protein extracts prepared from DS and DR cell membranes at different protein concentrations were reconstituted with S49CYC- membranes. The reconstituted S49CYC- membranes were isolated, solubilized, and analyzed for Gs{alpha} content by Western blot analysis as described in Materials and Methods. As determined from densitometric analysis, the levels of Gs{alpha} in S49CYC- membranes reconstituted with G protein extracts from DS and DR cells did not differ significantly (P > 0.05).

 
S49CYC- membranes reconstituted with G protein extracts from DR cells were more sensitive to Gpp(NH)p and less dependent on NaCl than were membranes reconstituted with extracts from DS cells. Gpp(NH)p, at 50 µM, was sufficient to shift all of the ß2ARs to low affinity in S49CYC- membranes reconstituted with DR extracts (Fig. 6Go). In addition, NaCl was not required for this effect of Gpp(NH)p (data not shown). In contrast, a higher concentration of Gpp(NH)p (200 µM) together with NaCl (120 mM) were required to shift receptors to low affinity in S49CYC- membranes reconstituted with DS extracts (Fig. 6Go). In the absence of NaCl, 200 µM Gpp(NH)p failed to shift receptors to a low affinity state (data not shown). Therefore, the distinctive effects of Gpp(NH)p and NaCl on agonist binding to ß2ARs associated with DR cells were transferred to S49CYC- membranes upon reconstitution with the DR extracts.



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  		Figure 6. Agonist displacement curves using membranes from reconstituted S49CYC- cells. ß2ARs were analyzed in S49CYC- membranes reconstituted with G protein extracts from DS (A) and DR (B) cells in a competition binding assay using [125I]iodocyanopindolol (ICYP) as the labeled ligand and isoproterenol as the unlabeled competitor. Binding analyses were performed on membranes with 0 µM Gpp(NH)p ({square}), 50 µM Gpp(NH)p plus 120 mM NaCl (•), or 200 µM Gpp(NH)p plus 120 mM NaCl ({triangleup}) as indicated. The results shown are representative of at least three independent experiments.

 
Sequence of Gs{alpha} cDNA from DS and DR cells
Gs{alpha} cDNA was cloned from parent DS and mutant DR cells on three overlapping fragments using a combination of reverse transcriptase-PCR and 5'-RACE techniques (Fig. 7AGo) and was subjected to DNA sequence analysis. For DS and DR cells, identical sequences (Fig. 7BGo) were obtained for the long form of Gs{alpha} (14), which represented the major isoform recovered from each cell line. The cDNA sequence differed at five positions from that reported previously for Gs{alpha} from wild-type S49 mouse lymphoma cells (Fig. 7bGo) (29); these differences resulted in two codon changes: a D139N substitution that also is seen in Gs{alpha} from a mouse macrophage cell line (20), and a conservative L198V substitution. Gs{alpha} cDNAs representing other splice variants of Gs{alpha} described previously in a human brain cDNA library (30) also were detected in DS and DR cells. These variants arose from alternate splicing events around the same intron-exon boundaries and were present in lower abundance.



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  		Figure 7. Gs{alpha} cDNA from mouse adrenocortical tumor cells. A, Total RNA was extracted from parental DS and mutant DR cells and used to prepare Gs{alpha}-specific cDNA (upper line) as detailed in Materials and Methods. The thick line segment represents the coding region of Gs{alpha}, whereas the thin line segments denote 5'- and 3'-untranslated regions. The Gs{alpha} cDNA was amplified on two overlapping fragments (middle lines) using forward and reverse primers (filled squares); the remainder of the Gs{alpha} transcript was amplified by 5'-RACE (lower line) using a Gs{alpha}-specific primer (filled square). B, The cDNA sequence and deduced amino acid sequence of the predominant isoform of Gs{alpha} obtained from DS and DR cells are shown. 5'- and 3'-untranslated sequences are represented by lowercase letters, and the location of the splice sites that generate this isoform of Gs{alpha} are marked ({blacktriangledown}). Differences from the cDNA and amino acid sequences reported for Gs{alpha} from S49 lymphoma cells (29) are indicated in bold and are underlined.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
As has been discussed extensively elsewhere (31, 32), the ability of G proteins to form functional complexes with receptors can be estimated by measuring the fraction of receptors that bind agonists with high affinity and that are converted to low affinity binding states in the presence of GTP analogs such as Gpp(NH)p. We evaluated these parameters of G protein function and found that different concentrations of guanyl nucleotide and NaCl were required to uncouple G proteins from their receptors in parent DS and mutant DR cells. The G proteins from DR cells uncoupled from their receptors at lower concentrations of Gpp(NH)p and in the absence of NaCl, whereas G proteins from DS cells required higher concentrations of Gpp(NH)p to uncouple from their receptors and were dependent upon NaCl. These differences in G protein function could be transferred to Gs{alpha}-deficient S49CYC- lymphoma cell membranes in reconstitution assays using extracts from parent DS or mutant DR cells, supporting the hypothesis that the mutant phenotype was associated with alterations in G protein activity.

The ability of Gpp(NH)p to shift receptors from a high affinity binding state to a low affinity state seems to reflect guanyl nucleotide exchange on Gs{alpha} (33). The occupancy of Gs{alpha} with GTP or GTP analogs is thought to destabilize receptor-Gs{alpha} interactions and enhance receptor-arrestin interactions, which, in turn, disrupt coupling between receptor and Gs (34). The different requirements for Gpp(NH)p and NaCl in DS and DR cells thus may reflect differences in guanyl nucleotide exchange rates on Gs{alpha}. Cloning and sequencing experiments demonstrated that DS and DR cells both expressed transcripts corresponding to the long form of Gs{alpha} without evidence of mutation. The absence of a Gs{alpha} mutation in DR cells suggests that additional factors contribute to guanyl nucleotide exchange and that the activity of one of these factors is altered in DR mutant cells. This factor is not likely to be ßAR kinase or ß-arrestin, because the DR mutation affects heterologous desensitization, which is independent of ßAR kinase or ß-arrestin (12), as well as homologous desensitization, which is dependent upon this pathway (11). Indeed, we find that ßAR kinase activity and ß-arrestin levels are similar in DS and DR cells (unpublished observations). On the other hand, G protein ß- and {gamma}-subunits, which are known to influence guanyl nucleotide exchange on Gs{alpha}, remain as possible candidates for the DR mutation. It may be possible to isolate the factor responsible for altered G protein activity using reconstituted S49CYC- membranes as an assay system to monitor activity during purification.

We have yet to demonstrate that the altered sensitivity of mutant DR cells to Gpp(NH)p and NaCl is causally linked to the mutation that results in desensitization resistance. In previous studies, we showed that DR cells not only desensitized more slowly than DS cells but recovered from desensitization more rapidly (10). Conceivably, increased guanyl nucleotide exchange in Gs{alpha} could enhance Gs{alpha}-GTP cycling and thereby favor receptor-G protein coupling over uncoupling interactions of receptor with arrestin.


   Acknowledgments
 
We thank Dr. S. Mumby (University of Texas Southwest Medical Branch, Dallas, TX) for antibodies to Gs{alpha}, and Dr. J. Northup (NIH, Bethesda, MD) for purified Gs.


   Footnotes
 
1 This work was supported by research grants from the Medical Research Council of Canada and the Canadian Cystic Fibrosis Foundation (to B.P.S.) and from the Natural Sciences and Engineering Research Council of Canada (to J.M.). Back

2 Supported by a studentship award from the Canadian Cystic Fibrosis Foundation. Back

Received August 21, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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