INTRODUCTION

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Heterotrimeric guanine nucleotide-binding proteins (G proteins)¹ couple heptahelical receptors to intracellular effectors and are composed of three subunits (, , and ), which are the products of separate genes (reviewed in Refs. 1-3). Each G protein is defined by its -subunit, which binds guanine nucleotide and couples to downstream effectors such as adenylyl cyclase and phospholipase C. The inactive GDP-bound -subunit is associated with a tightly but noncovalently bound -dimer. Upon activation by receptor, the -subunit undergoes a conformational change resulting in release of GDP with concomitant binding of GTP and dissociation from . The GTP-bound -subunit can then interact with and modulate specific effector enzymes or ion channels. For G_s, these include the stimulation of adenylyl cyclase and modulation of ion channels (4, 5). Hydrolysis of bound GTP to GDP by an intrinsic GTPase activity returns the -subunit to the inactive state. G protein -subunits can also be activated by incubation with GTPS, a nonhydrolyzable GTP analogue, or AlF₄, which binds to GDP-bound -subunits and mimics the -phosphate of GTP.

X-ray crystal structures of two G protein -subunits (transducin and Gi1) reveal that -subunits have two domains, a ras-like GTPase domain encoding the structural elements for guanine nucleotide binding and effector interaction and a highly helical domain that may be critical to prevent release of GDP in the inactive state (6-12). The guanine nucleotide resides in a cleft between the two domains, and it is thought that GDP release is effected by receptor-induced opening of the cleft. Comparative analysis of the crystal structure of inactive (GDP-bound) to that of activated (GTPS- or AlF₄-bound) -subunits demonstrates that the active conformation is attained by the movement of three regions (named switch 1, 2, and 3). The movement of switches 1 and 2 upon GTP binding is in direct response to the presence of the -phosphate group. Switch 3 has no direct contact with bound guanine nucleotide. Upon activation, switches 2 and 3 move toward each other and form multiple interactions. In transducin, switch 3 residues were shown to be critical for effector activation, possibly by conformational coupling with switch 2 (13). Switch 3 may also make contacts with the helical domain, which are important for high affinity guanine nucleotide binding (10).

Albright hereditary osteodystrophy (AHO) is an autosomal dominant disorder characterized by obesity, short stature, subcutaneous ossifications, and, in some cases, mental retardation. Most cases are associated with heterozygous inactivating mutations of the gene encoding G_s (14, 15). Within AHO kindreds, patients may have the somatic features of AHO alone (termed pseudopseudohypoparathyroidism (PPHP)) or AHO in association with resistance to multiple hormones that activate G_s-coupled signaling pathways (termed pseudohypoparathyroidism type Ia). While most mutations associated with AHO are frameshift deletions or splice junction mutations, some encode missense mutations that have specific effects on the functional properties of the G_s protein. Examples include mutations that affect receptor coupling (16), guanine nucleotide binding (17, 18), and activation (19).

In the present report, we describe a novel G_s missense mutation from an AHO patient in which an arginine residue in switch 3 (Arg²⁵⁸)² is substituted with tryptophan. Arg²⁵⁸ is a nonconserved residue adjacent to a highly conserved glutamic acid residue (Glu²⁵⁹) that is important for contact between switch 2 and 3 in the activated state (7, 12). We present evidence that substitution of Arg²⁵⁸ leads to defective GDP binding, resulting in increased thermolability and decreased activation by AlF₄. This mutation also leads to decreased receptor activation. In the crystal structure of G_s in the active state, Arg²⁵⁸ associates with a residue (Gln¹⁷⁰) located between the D and E helices of the helical domain, forming a "lid" over the guanine nucleotide binding pocket. Mutation of Arg²⁵⁸ is predicted to disrupt these interactions as well as an interaction between Asp¹⁷³ in the helical domain and Lys²⁹³ in the GTPase domain previously shown to be important for receptor- and AlF₄-induced activation (20). Analysis of this mutation suggests that switch 3 residues, in addition to being involved in the activation mechanism, are also involved in maintaining the basal state (i.e. sustaining GDP in the guanine nucleotide binding site).

	EXPERIMENTAL PROCEDURES

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Patient-- The patient was a 24-year old white male with a diagnosis of AHO and PPHP. His birth weight was 6 pounds, 11 ounces. By age 10 months, developmental delay, brachycephaly, and decreased muscle tone were noted. Throughout childhood he was small for his age and had a stocky appearance. By 6 years, learning disabilities as well as impulsive and aggressive behavior were noted, and over the past 2 years the patient has demonstrated increased compulsive behavior. During evaluation at NIH in 1995, the patient was noted to have short stature (62 inches), moderate obesity, and a rounded face with mildly depressed nasal bridge. The patient had brachydachtyly involving the distal phalanx of the first digit and the fourth metacarpals bilaterally and intracranial calcifications in the globus pallidus. Laboratory evaluation revealed no evidence for resistance to parathyroid hormone or thyrotropin. Both of the patient's parents as well as his two sisters are clinically unaffected. The patient consented to protocols approved by the NIDDK/NIAMS institutional review board.

Preparation of Erythrocyte Membranes and Determination of G_s Activity and G_s Expression-- Erythrocyte membranes were prepared from the patient, his parents, and three normal subjects as described previously (21) and frozen in aliquots at 70 °C. Erythrocyte membranes were reconstituted with membranes from mutant cyc S49 mouse lymphomas cells (which lack G_s expression), and adenylyl cyclase activity in the presence of isoproterenol (10 µM) and GTP (10 µM) was determined as described previously (21). Protein was determined by the method of Bradford (Bio-Rad). Immunoblots were performed using affinity-purified RM antibody (2 µg/ml), directed to the carboxyl-terminal decapeptide of G_s (22), and bands were quantified with a PhosphorImager (Molecular Dynamics, Inc.) as described previously (18).

Nucleic Acid Isolation and Genetic Analysis-- Genomic DNA was isolated from whole blood and screened for mutations within the gene encoding G_s (GNAS1) using PCR and temperature gradient gel electrophoresis (15, 23). PCR fragments were purified using Centricon 100 filters (Amicon) and directly sequenced using the Sequenase kit (U.S. Biochemical Corp.). RNA was isolated from 100-µl aliquots of whole blood, and RT-PCR was performed as described previously (18). For amplification of the GNAS1 exon 10 coding region, the primers were as follows: upstream, 5'-ATGTTTGACGTGGGTGGCCAGC-3'; downstream, 5'-CACAGAGATGGTGCGCAGCCAT-3' (24). The mutation was confirmed by digestion of PCR and RT-PCR products with the restriction enzyme MspI.

Construction of G_s Plasmids and in Vitro Transcription/Translation-- To generate G_s R258W, RT-PCR fragments amplified from the patient's RNA were digested with HincII and Sse8387I and ligated into the transcription vector pBluescript II SK (Stratagene, La Jolla, CA) that contained wild type human G_s cDNA (splice variant G_s-1, Ref. 24) from which the same HincII-Sse8387I restriction fragment had been removed. A control plasmid was created using the identical approach with material obtained from a normal control. G_s R258A (CGG to GCG) was generated by PCR using a mutagenic primer. The primers were as follows (1 µM each): upstream, 5'-GACAAAGTCAACTTCCACATGTTTGACGTGGGTGGCCAGCGCGATGAACG-3'; downstream (mutagenic), 5'-GAGCCTCCTGCAGGCGGTTGGTCTGGTTGTCCTCCGCGATGACCATGTTG-3'. The DNA template was linearized vector containing the wild type G_s cDNA (0.2-1 µg/ml). The PCR mixtures were denatured at 94 °C for 1 min, annealed at 65 °C for 1 min, and extended at 72 °C for 30 s for 20 cycles. PCR products were digested with HincII and Sse8387I and ligated into the pBluescript II SK containing G_s as described above. Mutations were verified by DNA sequencing, and synthesis of full-length G_s from each construct was confirmed by immune precipitation of in vitro translated products with RM antibody. In vitro transcription/translation was performed on G_s plasmids as described previously (18) using the TNT coupled transcription/translation system from Promega, with the exception that in most experiments no RNase inhibitor was added. We have found that deletion of this component reduces nonspecific initiation from downstream Met codons with no detectable loss of full-length G_s.

Adenylyl Cyclase Assays-- Wild type and mutant G_s in vitro transcription/translation products (10 µl of translation medium) were reconstituted into 25 µg of purified S49 cyc plasma membranes and tested for stimulation of adenylyl cyclase in the presence of various agents as indicated in Table I (18, 25). Reactions were incubated for 15 min at 30 °C, and the amount of [³²P]cAMP produced was measured as described previously (26). Background activities were determined from mock transcription/translation reactions and were subtracted for final presentation of the data. Data were normalized to the relative amount of G_s synthesis as determined by quantitation of [³⁵S]methionine-labeled in vitro transcription/translation products.

Trypsin Protection Assays-- Limited trypsin digestion of in vitro translated G_s was performed as described previously (18). Briefly, 1 µl of in vitro translated [³⁵S]methionine-labeled G_s was incubated in incubation buffer (20 mM HEPES, pH 8.0, 10 mM MgCl₂, 1 mM EDTA, 1 mM DTT) with or without 100 µM GTPS or 10 mM NaF/10 µM AlCl₃ at various temperatures for 1 h and then digested with 200 µg/ml tosylphenylalanyl chloromethyl ketone-treated trypsin for 5 min at 20 °C. In some experiments, GDP was also included in the preincubation. Reactions were terminated by boiling in Laemmli buffer. Digestion products were separated on 10% SDS-polyacrylamide gels, and the amount of 38-kDa protected fragment was measured by PhosphorImager analysis. The percentage of protection is the signal in the 38-kDa protected band divided by the signal in the undigested full-length G_s band times 100. For experiments examining the time course of GDP release, [³⁵S]methionine-labeled in vitro translates were incubated with 2 mM GDP for 1 h at 30 °C; chilled on ice and diluted 20-fold into incubation buffer with 100 µM GTPS; and then transferred to a 30 °C water bath. At the indicated times, aliquots were removed, and trypsin and GDP were added to attain final concentrations of 200 µg/ml and 1 mM, respectively. For the zero time point, trypsin was added prior to transfer to 30 °C. After the addition of trypsin, the samples were immediately placed in an ice water bath and incubated for 1 h. GTPS binds to a negligible degree within 1 h at 0 °C (data not shown).

Sucrose Density Gradient Centrifugation-- [³⁵S]methionine-labeled G_s was synthesized, and rate zonal centrifugation was performed on linear 5-20% sucrose gradients (200 µl) as described previously (18, 27). Gradients were prepared in 20 mM HEPES, pH 8.0, 1 mM MgCl₂, 1 mM EDTA, 1 mM DTT, 100 mM NaCl, 0.1% Lubrol-PX, and additions as described in the figure legend. 6-µl fractions were obtained and analyzed by SDS-polyacrylamide gel electrophoresis, and the relative amount of G_s in each fraction was quantified as described previously (18). G was isolated from bovine brain (28).

Expression and Purification of G_s from E. coli-- Plasmid pQE60 containing the long form of bovine G_s cDNA with a hexahistidine extension at the carboxyl terminus was a generous gift of A. G. Gilman and R. K. Sunahara. The Arg²⁵⁸ residue was mutated by site-directed mutagenesis using the Quickchange kit (Statagene). After mutagenesis, each cDNA was sequenced to confirm the presence of the desired mutation and to rule out PCR artifacts. After transformation into E. coli strain JM109, cultures were grown, G_s expression was induced, and cleared lysates were prepared as described previously (29). His-tagged G_s proteins were purified on 2.5-ml Ni²⁺-nitrilotriacetic acid resin columns (Qiagen) equilibrated with TP buffer (50 mM Tris-HCl, pH 8.0, 20 mM -mercaptoethanol, and 0.1 mM phenylmethylsulfonyl fluoride). Cleared lysates were loaded onto each column, and then each column was washed with 25 ml of TPG (TP buffer supplemented with 50 µM GDP) containing 500 mM NaCl, followed by 40 ml of TPG containing 50 mM NaCl and 10 mM imidazole. G_s was eluted with TPG containing 50 mM NaCl, 150 mM imidazole, and 10% glycerol and then exchanged into 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 2 mM DTT, 50 µM GDP, and 10% glycerol and stored at 80 °C at greater than 1.5 mg/ml.

Guanine Nucleotide Binding Assays-- Assays measuring the rate of binding of GTPS were performed as described previously (30). Briefly, 25 nM purified G_s was incubated with 1 µM [³⁵S]GTPS (~30,000 cpm/pmol) in 25 mM HEPES, pH 8.0, 1 mM EDTA, 100 mM NaCl, 10 mM MgCl₂, 1 mM DTT, and 0.01% Lubrol-PX in a final volume of 2 ml. At various times, 50-µl aliquots were removed and diluted with 2 ml of ice-cold stop solution (25 mM Tris-HCl, 100 mM NaCl, 25 mM MgCl₂, and 100 µM GTP) and maintained on ice until all samples were collected. Samples were then filtered under vacuum through nitrocellulose filters (Millipore Corp.), washed twice with 10 ml of stop solution without GTP, and dissolved in 10 ml of scintillation mixture. k_app values for GTPS binding were calculated using GraphPad Prism software.

	RESULTS

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Identification of G_s Biochemical and Genetic Defect in an AHO Patient-- The patient had somatic features of AHO but no evidence of hormone resistance. G_s bioactivity in the patient's erythrocyte membranes measured by the cyc reconstitution assay in the presence of isoproterenol (10 µM) and GTP (10 µM) was 52% of that present in normal subjects. G_s expression in these same membranes was 50% of normal based upon quantitative immunoblotting. The patient's mother and father, who do not have clinical evidence of AHO and who do not have a GNAS1 mutation (see below) showed no decrease in G_s bioactivity or G_s expression (data not shown). The GNAS1 gene was screened for mutations by temperature gradient gel electrophoresis analysis of PCR-amplified genomic DNA fragments encompassing GNAS1 exons 2-13 and their intron-exon splice junctions (15, 24). Temperature gradient gel electrophoresis analysis of a genomic fragment encompassing GNAS1 exons 10 and 11, which was amplified from the patient's genomic DNA, revealed abnormally migrating bands that were not present in either parent's sample or in numerous other normal control or patient samples (data not shown). By direct sequencing of the genomic DNA fragment (Fig. 1), the patient was shown to have a heterozygous single base substitution (C to T) within the coding region of exon 10 that encodes the substitution of tryptophan for arginine at codon 258 (G_s R258W). Arg²⁵⁸ is within the switch 3 region of G_s (7). This mutation destroys an MspI restriction site. Digestion of PCR-amplified genomic DNA fragments with MspI confirmed the presence of the mutation in the patient and its absence in either parent (data not shown). This therefore represents a de novo mutation within this kindred.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Direct sequencing of the exon 10 and 11 genomic fragment. Genomic DNA was amplified by PCR and directly sequenced. The oligonucleotide primers used for PCR were 5'-AAGAATTCTTAGGGATCAGGGTCGCTGCTC-3' (upstream primer) and 5'-CGCCCGCCGCGCCCCGCGCCCGTCCCGCCGCCCCCGCCCCATGAACAGCCAGCAAGAGTGGA-3' (downstream primer with GC clamp) with the underlined sequences complementary to the GNAS1 gene (15, 24). Whereas the direct sequence of genomic DNA amplified from a normal subject (left) reveals only a CGG triplet at codon 258 (arginine), the corresponding sequence in the affected patient (PPHP, right) reveals a T and C at the third position of the codon. This indicates a heterozygous single base substitution that encodes the substitution of tryptophan (TGG) for arginine (CGG) at codon 258 (24).

View larger version (37K): [in this window] [in a new window]   Fig. 2.   MspI digestion of RT-PCR products. A 235-base pair RT-PCR product spanning exon 10 was amplified from whole blood RNA. The products were digested with MspI and electrophoresed on a 6% polyacrylamide gel. The pedigree is shown above, with the filled square representing the affected patient and the white square and circle representing the proband's father and mother, respectively. The fragment size in base pairs is indicated on the left. The left lane contains undigested RT-PCR product (U). The patient has 235-, 135-, and 100-base pair bands, demonstrating the presence of mutant (undigested) and wild type (digested) products. Both parents demonstrate complete MspI digestion, since they lack the mutation.

Substitution of G_s Arg²⁵⁸ Leads to Decreased Activation by Activated Receptor or AlF₄-- G_s R258W was cloned into the transcription vector pBluescript, and the in vitro transcription/translation products were compared with those of wild type G_s in various biochemical assays. Since the presence of an amino acid with a bulky hydrophobic side chain (tryptophan) may introduce nonspecific steric effects, we also generated and analyzed an additional mutant in which Arg²⁵⁸ was replaced by alanine (G_s R258A). After reconstitution of translation products into purified S49 cyc membranes, each mutant was efficient at stimulating adenylyl cyclase in the presence of GTPS, with the response from G_s R258W about the same as and that from G_s R258A slightly greater than that of wild type G_s (see Table I). In contrast, both mutants had markedly decreased ability to stimulate adenylyl cyclase in the presence of isoproterenol or AlF₄ (Table I). Therefore, substitution of Arg²⁵⁸ leads to specific defects in activation by activated receptor or AlF₄.

View this table: [in this window] [in a new window]   Table I Adenylyl cyclase stimulation by Gs mutants In vitro transcription/translation products were mixed with purified cyc membranes and assayed for adenylyl cyclase stimulation as described under "Experimental Procedures." Results are expressed as the mean ± S.D. (n-1) of triplicate determinations and are corrected for the relative level of synthesis of each mutant to wild type. Gs R258W and Gs R258A were synthesized to 86 ± 17% (n = 16) and 105 ± 25% (n = 6) of wild type Gs levels as determined by in vitro translation with [35S]methionine, SDS-polyacrylamide gel electrophoresis, and PhosphorImager analysis. Background values determined from mock transcription/translation reactions (in pmol of cAMP/ml of translation medium/15 min as follows: GTP, 29 ± 1; isoproterenol, 39 ± 5; GTPS, 39 ± 2; and AlF4, 64 ± 6) were subtracted from each determination. Values are shown in pmol of cAMP/ml of translation product/15 min, and the percentage of the wild type value is shown in parentheses.

View larger version (41K): [in this window] [in a new window]   Fig. 3.   Trypsin protection of in vitro translated Gs R258W and R258A in the presence of GTPS or AlF4. In vitro translates were digested with tosylphenylalanyl chloromethyl ketone-treated trypsin (200 µg/ml) for 5 min at 20 °C after 1-h preincubations at various temperatures and in the presence of various agents indicated below. For each form of Gs, the full-length undigested Gs is shown in the far left lane, and complete digestion in the absence of activators is demonstrated in the next lane. Digestion of wild type Gs in the presence of either AlF4 or GTPS (100 µM) produced a 38-kDa protected band at all temperatures examined (for GTPS, only the results at 37 °C are shown, far right). On shorter exposures, it is clear that the 38-kDa protected band is actually a doublet (not shown). GTPS was able to protect both mutants at all temperatures. For Gs R258A, protection by AlF4 was normal at 30 °C and somewhat decreased at 37 °C, while for Gs R258W, protection by AlF4 was somewhat decreased at 30 °C and markedly decreased at 37 °C. For both mutants, the addition of excess GDP (2 mM) to the preincubation was able to partially or fully restore protection by AlF4. Quantitation of trypsin protection assays is presented in Table II.

View this table: [in this window] [in a new window]   Table II Influence of excess GDP on AlF4-induced trypsin protection

Substitution of G_s Arg²⁵⁸ Leads to Decreased Affinity for GDP-- GTPS was a more effective activator of both mutants than AlF₄ at 37 °C. It is possible that both mutants have decreased affinity for AlF₄ or activate poorly when GDP and AlF₄ are bound. Another possibility is that at higher temperatures the mutants bind GDP more poorly, which would result in decreased activation by AlF₄, since GDP binding is a prerequisite for AlF₄ binding and activation. To address these possibilities, the effect of increasing the concentration of GDP on AlF₄-induced trypsin protection of G_s R258W and R258A was determined³ (Fig. 3, Table II). In the presence of increased GDP concentrations, the ability of each mutant to be protected by AlF₄ was partially or fully restored to normal. The effect was dose-dependent with maximum protection attained at a GDP concentration of 2 mM (data not shown). These data suggest that decreased protection of the mutants by AlF₄ is not due to a specific defect in AlF₄ binding but rather to decreased ability of the mutants to maintain the GDP-bound state at higher temperatures. GTP was equally effective in enhancing protection in the presence of AlF₄ (data not shown). In the absence of AlF₄, no protection was observed with either GDP or GTP. The latter observation suggests that the mutants have intact GTPase function.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Time course of GTPS-induced trypsin resistance. In vitro translates were incubated with 2 mM GDP for 1 h at 30 °C and then chilled on ice and diluted 20-fold into a solution containing 100 µM GTPS and then transferred to a 30 °C water bath. At the indicated times, aliquots were removed and mixed with a solution of tosylphenylalanyl chloromethyl ketone-treated trypsin and GDP (final concentrations, 200 µg/ml and 1 mM, respectively). For the zero time point trypsin was added prior to transfer to 30 °C. After the addition of trypsin, the samples were immediately placed in an ice water bath and incubated for 1 h. GTPS binds to a negligible degree within 1 h at 0 °C. Trypsin digestion was terminated, and the percentage of trypsin protection was determined as described under "Experimental Procedures." The data presented are the mean ± S.D. (n  1) of three independent experiments. The data for wild type Gs () are the same in the top and bottom panels. The data for Gs R258A (, top panel) and Gs R258W (; bottom panel) were separated for clarity. For both mutants, the apparent rate of GDP release (equivalent to the rate of increase of trypsin protection in the presence of GTPS) was at least 4-5 times the rate for wild type Gs.

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View larger version (18K): [in this window] [in a new window]   Fig. 5.   Time course of GTPS binding to purified Gs. Bovine wild type Gs and Gs R258A with carboxyl-terminal hexahistidine extension were expressed and purified from E. coli, and the rate of GTPS binding for each was determined. Wild type Gs () and Gs R258A () were incubated in 1 µM [35S]GTPS (~30,000 cpm/pmol) at 20 °C in 25 mM HEPES, pH 8.0, 1 mM EDTA, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, and 0.001% Lubrol-PX. At the indicated times, the reaction was terminated, and bound GTPS was determined as described under "Experimental Procedures." Data were fit (r2 > 0.995) to the equation B = Bmax(1  e), where B represents GTPS bound at time t, Bmax represents maximal GTPS bound, and k represents the apparent on rate. Each point is the mean ± S.D. of triplicate determinations. The Bmax values were 3.0 pmol for wild type Gs and 1.3 pmol for Gs R258A. kapp values averaged from three independent experiments were 0.04 ± 0.00 min1 for wild type Gs and 0.36 ± 0.02 for Gs R258A.

Decreased GDP Binding by G_s Arg²⁵⁸ Mutants Increases Their Thermolability-- Since the rate of GDP release from the G_s Arg²⁵⁸ mutants is faster than that from wild type G_s, we would predict that a greater proportion of each mutant exists in the guanine nucleotide-free state. Other G_s mutants with decreased affinity for guanine nucleotide have been shown to have increased thermolability (17, 18), presumably since the guanine nucleotide-free state is unstable. To examine the thermostability of G_s R258A and R258W, we analyzed their distribution within sucrose gradients after preincubation at various temperatures for 1 h in the presence or absence of 2 mM GDP³ or 100 µM GTPS (Fig. 6A). When in vitro translates of G_s R258A and R258W were held on ice, the gradient profile was virtually the same as that of wild type G_s and consistent with the overall proper conformation (sedimentation coefficient ~3.7 S; Ref. 18). When preincubated on ice with purified bovine brain , the sedimentation coefficient of both mutants and wild type G_s increased from 3.7 to 5.0 S (Fig. 6B), demonstrating that at low temperatures each mutant maintained the ability to interact with (18). Similar results were obtained with G_s R258W after incubation with at 30 °C (data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Sucrose density gradient centrifugation of Gs in vitro translation products. A, [35S]methionine-labeled in vitro translates of both Gs Arg258 mutants and wild type Gs were preincubated for 1 h at 0 (), 30 (), 37 (), or 37 °C in the presence of an added 2 mM GDP () or 37 °C in the presence of added 100 µM GTPS (). Following incubation, the samples were layered over a 200-µl 5-20% linear sucrose gradient and centrifuged for 1 h at 4 °C at 436,000 × g. Fractions (6 µl each) were collected, and odd numbered fractions were analyzed by SDS-polyacrylamide gel electrophoresis and PhosphorImager analysis (18). The data are expressed as the percentage of total Gs present in each fraction. Fraction 1 represents the top of the gradient. For samples in which either GDP or GTPS was included in the preincubation, it was also present in the gradient itself. The positions of the peak concentrations of standard proteins in the gradient are shown at the top and are as follows: soybean trypsin inhibitor, 2.3 S; carbonic anhydrase, 2.7 S; ovalbumin, 3.5 S; bovine serum albumin, 4.4 S, and phosphorylase b, 8.4 S. B, [35S]methionine-labeled in vitro translates of both Gs Arg258 mutants and wild type Gs were preincubated for 1 h at 0 °C in the presence or absence of purified bovine brain G (20 µg/ml) and subjected to sucrose density gradient centrifugation. In the presence of , both mutants and wild type Gs shifted to a peak of ~5 S. For clarity, each mutant is shown only in the presence of .

	DISCUSSION

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We identified a heterozygous missense mutation (G_s R258W) in a patient with AHO and PPHP. This mutation, as well as a mutation that replaces Arg²⁵⁸ with alanine (G_s R258A), encodes a G_s protein with impaired function. Both mutants stimulated adenylyl cyclase and attained the active conformation normally in the presence of GTPS. However, their ability to stimulate adenylyl cyclase in the presence of AlF₄ or activated receptor (isoproterenol plus GTP) was significantly attenuated. Consistent with this result, GTPS was better able than AlF₄ to protect both mutants from trypsin digestion. Excess GDP was able to partially or fully restore AlF₄ protection, suggesting that decreased activation by AlF₄ was due to decreased binding of GDP in the inactive state. Both mutants were shown to have a markedly increased rate of guanine nucleotide turnover, reflecting an increased rate of GDP release. Defective guanine nucleotide binding in these mutants probably results from disruption of interactions between the helical and GTPase domains (see below). Although the overall conformation (and ability to bind ) of both mutants was normal at lower temperatures, both denatured more rapidly at physiological temperatures. The mutants were protected from denaturation by excess guanine nucleotide, suggesting that increased thermolability is the direct result of decreased guanine nucleotide binding. Decreased guanine nucleotide binding is associated with increased thermolability in other G_s mutations (17, 18), and it is presumed that -subunits are unstable in the absence of bound guanine nucleotide. The expression of G_s is significantly reduced in erythrocyte membranes from the affected patient and membrane targeting of G_s R258W is reduced when expressed in S49 cyc cells (data not shown). Whether abnormal targeting is due to denaturation or more subtle conformational changes affecting or membrane binding is unclear. However, lack of expression of G_s R258W in the membrane at physiological temperature is probably the overriding defect in the affected patient.

Although G_s R258W shows a somewhat more severe phenotype than G_s R258A, both essentially behave similarly, suggesting that substitution of Arg²⁵⁸ is disrupting specific interactions of this residue with other G_s residues. Examination of the molecular structure of GTPS-bound G_s reveals a direct contact between Arg²⁵⁸ (in switch 3 of the GTPase domain) with a residue (Gln¹⁷⁰) in the helical domain located in the loop between D and E (Fig. 7). Mutation of Arg²⁵⁸ to either tryptophan or alanine would be predicted to disrupt this interaction between the GTPase and helical domains. Since interactions between Arg²⁵⁸ and the helical domain act as a "lid" over the cleft that contains the guanine nucleotide binding pocket, mutations of Arg²⁵⁸ would be predicted to open the cleft and allow greater dissociation of the bound guanine nucleotide, as observed in our experiments. Arg²⁵⁸ is not conserved among different G protein -subunits but is conserved in G_s from various species. In contrast, Gln¹⁷⁰ is highly conserved, and in G_s it also interacts with Val²⁵⁶, which is conserved in most G protein -subunits. Substitution of Arg²⁵⁸ might also perturb the interaction between Gln¹⁷⁰ and Val²⁵⁶.

View larger version (40K): [in this window] [in a new window]   Fig. 7.   Crystal structure of Gs-GTPS. The coordinates for the short form of bovine Gs-GTPS (Protein Data Bank accession code 1AZT (12)) were loaded into Look, version 3.0. The numbering corresponds to the long form of Gs. The atoms in residue Arg258 in the GTPase domain and residue Gln170 in the helical domain are shown as space-filled CPK spheres (carbon, gray; oxygen, red; nitrogen, blue; sulfur and phosphorus, yellow). These interactions keep a "lid" over the cleft between the two domains. Asp173 in the helical domain and Lys293 in the conserved NKXD motif of the GTPase domain, which form a salt bridge between the two domains, are also highlighted. GTPS is shown in a yellow and red stick model, and the remainder of the protein is shown as the -carbon trace.

In G_s there is salt bridge between the side chains of Asp¹⁷³ in the helical domain and Lys²⁹³ in the GTPase domain (within the conserved guanine nucleotide binding motif NKXD) (Fig. 7). Codina and Birnbaumer (20) showed that mutating either residue⁴ results in normal activation by GTPS but decreased activation by AlF₄ or activated receptor, similar to what we observed with Arg²⁵⁸ substitutions. Since both Asp¹⁷³ and Gln¹⁷⁰ lie within the interhelical loop between D and E, which is in close contact with the GTPase domain, it is possible that mutations of Arg²⁵⁸ may also disturb the salt bridge between Asp¹⁷³ and Lys²⁹³, which would lead to defective activation. Repositioning of the conserved lysine in the NKXD motif could directly result in decreased guanine nucleotide binding by altering the conformation of the guanine nucleotide binding pocket.

The exact mechanism by which substitution of Arg²⁵⁸ leads to defective receptor-mediated activation is not well defined. Several regions in transducin that are critical for receptor binding and activation were identified by scanning mutagenesis, although the switch 3 region was not mutagenized in that study (37). Decreased receptor-mediated activation could be the direct result of decreased binding to or receptor. The Arg²⁵⁸ mutants were capable of binding to . Li and Cerione (13) demonstrated that deletion of the whole switch 3 region of transducin had no effect on interactions with or its receptor (rhodopsin). Moreover, crystal structures of transducin and G_i do not demonstrate direct interactions between switch 3 and (9, 11). Disturbance of the salt bridge between the helical and GTPase domains results in severely decreased receptor activation (see above; Ref. 20). Studies on G_s/G_i2 chimeras suggest that interactions between switch 3 residues and the helical domain are critical not only to maintain the basal state but also for receptor-mediated activation (38).

Decreased receptor activation could be the direct result of a GTP binding defect, as demonstrated by mutation of a conserved switch 2 arginine (G_s R231H) in an AHO patient (19, 39). Similar to the Arg²⁵⁸ mutations, this mutation leads to normal GTPS-mediated but decreased AlF₄- and receptor-mediated activation. In the GTP-bound state, Arg²³¹ interacts with several residues in the 3 helix and switch 3 regions, including the conserved glutamic acid residues Glu²⁵⁹ and Glu²⁶⁸. Disrupting these interactions presumably leads to a GTP binding defect, resulting in decreased receptor-mediated activation (19). It is of interest that mutation of Glu²⁵⁹ results in a similar phenotype.⁵ In contrast to the Arg²⁵⁸ mutants, G_s R231H did not appear to have a GDP binding defect. GTP alone did not protect either Arg²⁵⁸ mutant or wild type G_s from trypsin protection, but it was able to restore trypsin protection of G_s R258W in the presence of AlF₄ with a similar dose response as GDP (data not shown), suggesting that GTP as well as GDP binding may be altered by substitution of Arg²⁵⁸. While GTPS binding might also be predicted to be decreased, it has been proposed that defective GTP binding may result in a conditional activation defect that is only obvious in states in which guanine nucleotide binding is destabilized (such as interaction with activated receptor; Ref. 19). It was postulated that decreased activation of G_s R231H by AlF₄ is due to an inability of the mutant to stably maintain the GDP-AlF₄ complex in the guanine nucleotide binding pocket, and it was demonstrated that the complex could be stabilized by high concentrations of Mg²⁺ (19). The Arg²⁵⁸ mutants showed decreased activation by AlF₄ even in the presence of 10 mM Mg²⁺. Moreover the defect was corrected with high concentrations of GDP, suggesting that for G_s R258W and R258A, decreased activation by AlF₄ is due to decreased GDP binding.

In summary, genetic analysis of the gene encoding G_s in an AHO patient identified a residue in the switch 3 region that is critical for normal guanine nucleotide binding and receptor activation. This residue interacts with a residue in the helical domain and underscores the importance of interdomain interactions in both guanine nucleotide binding and receptor-mediated activation. This study demonstrates that identification and analysis of G_s mutations in AHO patients can further our understanding of G protein function.