Pseudohypoparathyroidism, a novel mutation in the betagamma-contact region of Gsalpha impairs receptor stimulation.

Pseudohypoparathyroidism, type Ia (PHP-Ia), is a dominantly inherited endocrine disorder characterized by resistance to hormones that act by stimulating adenylyl cyclase. It is caused by inheritance of an autosomal mutation that inactivates the α subunit (αs) of Gs, the stimulatory regulator of adenylyl cyclase. In three members of a family, the PHP-Ia phenotype is associated with a mutation (R231H) that substitutes histidine for an arginine at position 231 in αs. We assessed signaling function of αs-WT versus αs-R231H transiently transfected in HEK293 cells. Hormone receptor-dependent stimulation of cAMP accumulation in cells expressing αs-R231H is reduced by ∼75% in comparison to cAMP accumulation in cells expressing αs-WT. A second mutation, αs-R201C, inhibits the GTPase turnoff reaction of αs, thus producing receptor-independent stimulation of cAMP accumulation. The double mutant, αs-R231H/R201C, stimulates cAMP accumulation almost as well (∼80%) as does αs-R201C itself, indicating that the R231H mutation selectively impairs receptor-dependent signaling. In three-dimensional structures of G protein heterotrimers, Arg-231 is located in a region, switch 2, that is thought to interact with the βγ subunit rather than with the hormone receptor. Thus, the R231H phenotype suggests that switch 2 (perhaps in concert with βγ) mediates G protein activation by receptors at a site distant from the receptor-G protein contact surface.

both subunits to interact with effectors. Effector stimulation is then terminated by the intrinsic GTPase activity of G␣, followed by reassociation of G␣GDP and ␤␥. With respect to this cycle, recently published crystal structures have revealed details of GTP-induced conformational change in G␣ (1-3), a plausible structure for the catalytic intermediate in the GTPase reaction (4,5), and three-dimensional structures for the G␣␤␥ heterotrimer (6,7). Because we do not have the structure of a receptor-G␣␤␥ complex, however, we cannot yet describe in molecular detail the pivotal event in receptor-G protein signaling, receptor-triggered release of GDP from G␣.
At present we can try to understand this event by interpreting effects of instructive mutations. A potential source of such mutations is an inherited disease of G protein signaling, pseudohypoparathyroidism, type Ia (PHP-Ia) (8). PHP-Ia patients inherit a defect in one of the two autosomal alleles of the gene for ␣ s , the ␣ subunit of G s , the stimulatory regulator of adenylyl cyclase. Although G s mediates effects of many hormones, the ϳ50% decrease in G s activity produced by loss of an autosomal allele causes clinically evident impairments of responsiveness to two hormones, parathyroid hormone (PTH) 1 and thyroid stimulating hormone (TSH). Resistance to PTH causes hypocalcemia and hyperphosphatemia, while resistance to TSH produces hypothyroidism.
Since the discovery of G s deficiency in PHP-Ia (9, 10), investigators have reported a large number of ␣ s mutations in patients with this disorder (8). Unfortunately, most of these ␣ s mutations globally inactivated the protein (e.g. by premature chain termination) and therefore proved useless for understanding detailed mechanisms of G protein function. One PHP-Ia mutation appeared specifically to impair interaction of the mutant ␣ s with receptors, in keeping with the location of the mutation (R385H) at a site, near the G␣ C terminus, that was already known to interact with receptors (11). We characterized another ␣ s mutation, A366S, found in patients with a rare syndrome that combines testitoxicosis and PHP-Ia; in this case, the PHP-Ia phenotype resulted from thermal instability of the mutant ␣ s (12). Here we report a third PHP-Ia mutation, R231H. Because this mutation impairs responsiveness of ␣ s to receptor stimulation, its location at a site that interacts with ␤␥ rather than with receptor is of special interest.

EXPERIMENTAL PROCEDURES
Analysis of the ␣ s Gene-Using genomic DNA from peripheral blood leucocytes, exons 2-13 and their flanking intron sequences in the human ␣ s gene (13) were amplified by the polymerase chain reaction (PCR), as described previously (14). Amplified DNA fragments were sequenced directly, using a Promega fmol Sequencing Kit.
Plasmid Construction and Transfection-An ␣ s cDNA containing an internal hemagglutinin epitope, previously described (15), was used as a PCR template to introduce a histidine substitution for arginine at position 231 and (in other constructs) a cysteine substitution for arginine at position 201. The mutated region was ligated back into pcDNA1, and the sequence of the PCR-generated fragment was confirmed by dideoxy sequencing. HEK293 cells were propagated and transiently transfected exactly as described (16), using the DEAE-dextran method (17).
cAMP Assay-One day after transfection, cells were reseeded into wells of a 24-well plate, and [ 3 H]adenine (2 Ci/ml) was added. One day later, cells were washed in 0.5 ml of assay medium containing 1 mM * This work was supported in part from the United States-Israel Binational Science Foundation (to Z. F.) and the National Institutes of Health (to H. R. B.). 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.
Immunoblots-Proteins were resolved by 10% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and probed with monoclonal antibody 12CA5 as described (15). Immunoreactive proteins were specifically detected by incubation with horseradish peroxideconjugated sheep anti-mouse antibody (Amersham) and developed with enhanced chemiluminescence (Amersham).

RESULTS
Three of five family members showed clinical characteristics of PHP-Ia (Fig. 1, Table I), including the distinctive skeletal abnormalities of Albright's hereditary osteodystrophy, disturbed Ca 2ϩ homeostasis (elevated PTH, low Ca 2ϩ , and elevated PO 4 ), and hypothyroidism (elevated TSH and decreased thyroxine). The mother (patient I-1) had mild Albright's hereditary osteodystrophy and normal or only slightly abnormal endocrine function. Only the three clinically affected individuals (I-1, II-1, and II-3) showed the R231H mutation (substitution of an adenine for a guanine residue) in exon 9 of one ␣ s gene (Fig. 2).
To assess the ability of wild type or mutant ␣ s to mediate receptor-stimulated cAMP synthesis, we co-transfected the ␣ s with a porcine ␣2-adrenoreceptor (␣2-AR) and measured cAMP accumulation after stimulation with an ␣2-AR agonist, UK-14304, administered at 10 M (Fig. 3A) or in a concentrationresponse curve (Fig. 3B). In both cases, UK-14304 stimulated cAMP accumulation much more effectively in cells expressing exogenous ␣ s -WT than in cells transfected with vector alone; in cells expressing ␣ s -R231H, the ␣2-AR agonist also stimulated cAMP accumulation, to an extent slightly greater than observed with transfection of vector alone, but much (ϳ75%) less effectively than in cells expressing ␣ s -WT. From these results we infer that the R231H mutation markedly impairs ability of ␣ s to stimulate cAMP accumulation in response to receptor stimulation. As in the past (16), we measured cAMP accumulation in response to an agonist acting on the transiently transfected ␣2-AR, which stimulates G s quite weakly, rather than in response to a receptor that efficiently stimulates G s ; because the latter kind of receptor, but not the ␣2-AR, can efficiently stimulate the limited concentration of endogenous ␣ s , use of the ␣2-AR agonist allowed us to measure selectively the cAMP  Albright's hereditary osteodystrophy accumulation that depended predominantly on transiently transfected ␣ s (16). Note that the R231H defect was not associated with reduced expression of the mutant protein; indeed, immunoblots show that ␣ s -R231H was expressed at least as well as ␣ s -WT in these experiments (Fig. 3C).
The R231H defect observed in Fig. 3 could have resulted from intrinsic inability of the mutant ␣ s to stimulate adenylyl cyclase, rather than from a selective defect in ability to respond to receptor. Accordingly, we assessed ␣ s -dependent, receptorindependent cAMP accumulation after transfecting cells with ␣ s containing a second mutation, R201C; the R201C mutation reduces the GTPase activity of ␣ s , thereby preventing the GTPbound protein from turning itself off and making receptor stimulation unnecessary (19). In these experiments (Fig. 4), expression of ␣ s -R201C caused substantial cAMP accumulation, 3.3fold greater than seen with ␣ s lacking the GTPase-inhibiting mutation. The R201C/R231H double mutant stimulated cAMP accumulation almost as well as did ␣ s -R201C itself. We infer that the R231H mutation impairs ␣ s -adenylyl cyclase interaction only slightly, to an extent considerably less than its impairment of ␣ s -receptor interaction. DISCUSSION Arg-231 in ␣ s , which corresponds to Arg-204 in the ␣ subunit (␣ t ) of retinal transducin, is a highly conserved residue in the "switch 2" region, an ␣ helix (␣2) that interacts directly with the ␤␥ subunit and that also plays an important role in GTPdependent conformational change in G␣. By interacting with a main-chain amide at the N terminus of ␣2, the ␥-phosphate of GTP induces a change in orientation of ␣2 and a twist about its axis (1,2); in this conformation the side chain corresponding to Arg-231 binds a negatively charged glutamate in the "switch 3" loop. Thus, Arg-231 might be important for mediating the GTP-induced changes that cause dissociation from ␤␥ and al-low stimulation of effectors. Although the three-dimensional structures make it easy to imagine that a substitution of histidine at position 231 could impair effector stimulation, ␣ s -R231H in fact stimulates adenylyl cyclase almost as efficiently as does ␣ s -WT (Fig. 4).
Instead, we must ask how the R231H mutation could impair receptor-induced replacement of GDP by GTP. In the ␣␤␥ heterotrimer (6), which is likely to serve as the receptor target, the arginine in this position (Arg-204 in ␣ t ) is oriented toward the G␣ hydrophobic core rather toward the ␤␥ subunit, which interacts directly with the ␣2 residues on either side of the arginine. It is hard to see how a histidine at this position could interfere with the interaction of G protein and receptor, because no possible orientation of the receptor would allow it to contact Arg-204. Consequently, we are left with two possibilities. In the absence of ␤␥, receptors cannot release GDP from G␣; thus, it is possible that histidine at this position could block receptor activation by somehow impairing the interaction of G␣ with ␤␥. Alternatively, or in addition, the receptor may interact with the ␣2/␤4 loop at the C terminus of the ␣2 helix; thus, this helix could transmit conformational change from the receptor to the guanine nucleotide binding pocket, near the N terminus of ␣2, and histidine substituted for arginine at position 231 might block this conformational change.