Human heart failure is associated with a diminished contractile response to 0-adrenergic agonists. We hypothesized that alterations in the activity of a guanine nucleotide-binding regulatory protein (G protein) might be partially responsible for this abnormality. We therefore measured the activity of G proteins in failing human myocardium utilizing bacterial toxin-catalyzed ADP ribosylation. The activity of a 40,000-mol wt pertussis toxin substrate (aG40) was increased by 36% in failing human hearts when compared with nonfailing controls. In contrast, there was no change in the level of the stimulatory regulatory subunit (G.). The increased activity in aG40 was associated with a 30% decrease in basal as well as 5'-guanylyl imidodiphosphate-stimulated adenylate cyclase activity. These data suggest that increased aG40 activity is a new marker for failing myocardium and may account at least in part for the diminished responsiveness to 01-adrenergic agonists in the failing human heart.

We have localized a guanine nucleotide-bind-

Transducin, a GTP-binding protein involved in phototransduction in the vertebrate retina, belongs to a family of homologous coupling proteins that also includes G, and G1, the regulatory proteins of adenylate cyclase. Here we report the cDNA sequence and deduced amino acid sequence of transducin's a subunit (Ta). The cDNA was isolated, by screening with an antibody probe, from a bovine retinal cDNA library in the expression vector Xgtll. The 2.2-kilobase cDNA insert hybridized to a single 2.6-kilobase poly(A)+ RNA species present in extracts of bovine retina but not of bovine heart, liver, or brain. The nucleotide sequence of the cDNA revealed an open reading frame long enough to encode the entire 39-kDa T. polypeptide. The polypeptide sequence deduced from the cDNA would be composed of 350 amino acids and have a molecular weight of 39,971. Portions of the sequence matched reported amino acid sequences of Ta tryptic fragments, including sites specificlly ADP-ribosylated by cholera and pertussis toxins. The predicted sequence also includes four segments, ranging from 11 to 19 residues in length, that exhibit significant homology to sequences of GTP-binding proteins, including the ras proteins of man and yeast and the elongation factors of ribosomal protein synthesis in bacteria, EF-G and EF-Tu. In combination with previous functional studies of tryptic fragments of Ta, the deduced amino acid sequence makes it possible to predict which portions of the polypeptide interact with other molecules involved in retinal phototransduction.Transducin, a guanine nucleotide binding protein of retinal rod cells, mediates the activation of a cyclic GMP phosphodiesterase (PDEase) in response to photoexcitation of rhodopsin (1). Each photolyzed rhodopsin (R*) molecule activates hundreds of transducin molecules by catalyzing the exchange of GTP for GDP at transducin's guanine nucleotide binding site. The activated transducin-GTP complex in turn stimulates the hydrolysis of many cGMP molecules by PDEase. Hydrolysis of bound GTP returns transducin to its inactive state.Transducin contains three subunits, a, ,B, and y, whose sizes are 39, 35, and =8 kDa, respectively. Transducin exhibits striking structural and functional homologies to the stimulatory (Gs) and inhibitory (Gi) coupling proteins of hormone-sensitive adenylate cyclase. Each protein is a heterotrimer in which the respective subunits exhibit similar amino acid compositions and proteolytic peptide maps (2). In each case, the protein's interaction with an excited signal detector (rhodopsin or hormone receptor) triggers dissociation of the /3y complex from the a subunit, which then alters activity of the effector molecule (PDEase or adenylate cyclase) (1, 3).After hydrolysis of GTP bound to the a subunit, the P-y

Recently, a unique Pro250Arg point mutation in fibroblast growth factor receptor 3 (FGFR3) was reported in 61 individuals with coronal craniosynostosis from 20 unrelated families [Muenke et al. (1997): Am J Hum Genet 60:555-564]. The discovery of this apparently common mutation has resulted in the definition of a recognizable syndrome, through analysis of subtle clinical findings in families who were previously thought to have a variety of other craniosynostosis syndromes. Previous diagnoses in some of these families have included Jackson-Weiss, Saethre-Chotzen, and Pfeiffer syndromes, as well as Adelaide-type craniosynostosis and brachydactyly-craniosynostosis syndrome [Adès et al. (1994): Am J Med Genet 51:121-130; von Gernet et al. (1996): Am J Med Genet 63:177-184; Reardon et al. (1997): J Med Genet 34:632-636; Bellus et al. (1996): Nat Genet 14:174-176; Hollaway et al. (1995): Hum Mol Genet 4:681-683; Glass et al. (1994): Clin Dysmorphol 3:215-223]. There appears to be a need to further delineate the phenotype associated with this common mutation in FGFR3. We compare the clinical characteristics of previously reported cases of this unique Pro250Arg mutation with those of two additional families and suggest that this syndrome with a unique mutational basis be designated coronal craniosynostosis with brachydactyly and carpal/tarsal coalition due to Pro250Arg mutation in FGFR3 gene, to emphasize the distinctive findings which may be present even in the absence of coronal craniosynostosis.

Two of four siblings expressed the salt-losing form of congenital adrenal hyperplasia due to 21-hydroxylase deficiency (CAH) and had identical human lymphocyte antigen (HLA) and complement C4 (fourth component of complement) types (HLA-A3,C4,B35,C4A3,C4BQO,DR1/A2,C-,B18,C4A3, C4BQO,DR6). The father and one unaffected sibling were heterozygous carriers of CAH, as determined by a 30-min iv ACTH stimulation test and HLA typing. In addition, the iv ACTH stimulation test revealed that the mother and the other unaffected sibling also carried an allele for an attenuated form of CAH. Restriction endonuclease digests of genomic DNA obtained from members of this family and from normal unrelated subjects were hybridized with cDNA probes encoding human 21-hydroxylase and C4. With the 21-hydroxylase probe, Southern blots prepared from control DNA samples revealed two major restriction fragments in each of four restriction endonuclease digests; TaqI produced major bands at 3.7 and 3.2 kilobases (kb), KpnI at 4.0 and 2.9 kb, EcoRI at 18 and 13 kb, and BglII at 15 and 12.5 kb. Southern blots prepared from DNA of the two patients lacked the 3.7-kb TaqI and 2.9-kb KpnI fragments, but had increased hybridization intensity (relative to control DNA samples) in the 3.2-kb TaqI and 4.0-kb KpnI fragments. By contrast, blots with EcoRI or BglII had two large hybridization fragments not different from control DNA samples. These data indicate the presence of two different 21-hydroxylase genes. Additional mapping studies revealed that the two genes had the restriction pattern of the inactive 21-hydroxylase gene. When genomic DNA that had been isolated from all members of this family and from normal subjects was hybridized with the human C4 cDNA probe, the restriction fragment hybridization patterns for all four endonuclease digests were similar in the two groups. Hence, our results suggest that the 21-hydroxylase deficiency of our patients is due to conversion of the active 21-hydroxylase gene to the inactive gene. This gene conversion was associated with absence of functional C4B protein, without any detectable alterations in the restriction fragment pattern of the C4 genes.

Familial male precocious puberty (FMPP) is a gonadotropin-independent disorder that is inherited in an autosomal dominant, male-limited pattern. A heterozygous mutation encoding substitution of Asp578 with Gly in transmembrane helix 6 of the G protein-coupled receptor for luteinizing hormone (LHR) has been found in affected males from nine American FMPP families. Cells expressing the mutant LHR exhibit markedly increased cyclic adenosine monophosphate (cAMP) production in the absence of agonist, suggesting that autonomous Leydig cell activity in FMPP is caused by a constitutively activated LHR. We have now analyzed genomic DNA from affected males from six additional FMPP families. PCR was used to amplify a fragment of the LHR gene encoding amino acid residues 441-594. None of the six new samples contained the Asp578-->Gly mutation, as indicated by absence of digestion with MspI. PCR products were then screened for heterozygous mutations using temperature-gradient gel electrophoresis. DNA fragments from two of the patients migrated abnormally. Direct sequencing of PCR product from one affected German male revealed a heterozygous mutation (ATG-->ATA) encoding Met571-->Ile at the cytoplasmic end of helix 6, the same mutation that has been reported in another European FMPP kindred. Affected males in the second family had a novel Thr577-->Ile mutation (ACC-->ATC). Mutations in different portions of the LHR or in a different gene may be responsible for disease in the other FMPP kindreds. Agonist binding and functional coupling of the mutant receptors to the cAMP and inositol phosphate pathways were studied by transiently expressing them in COS-7 cells. Agonist affinity was unaffected by the mutations.(ABSTRACT TRUNCATED AT 250 WORDS)

Patients who have pseudohypoparathyroidism type I associated with Albright hereditary osteodystrophy commonly have a genetic deficiency of the a subunit of the G protein that stimulates adenylyl cyclase (aG3) (ATP pyrophosphate-lyase, EC 4.6.1.1). To discover the molecular mechanism that causes aG5 deficiency in these patients, we examined eight kindreds with one or more members affected with Albright hereditary osteodystrophy or pseudohypoparathyroidism and aG. deficiency. In these families, aG. deficiency and the Albright hereditary osteodystrophy phenotype were transmitted together in a dominant inheritance pattern.Using a cDNA hybridization probe for aG., restriction analysis with several endonucleases showed no abnormalities of restriction fragments or gene dosage. RNA blot and dot blot analysis of total RNA from cultured fibroblasts obtained from the patients revealed ==50% reduced mRNA levels for aGS in affected members of six of the pedigrees but normal levels in affected members of the two other pedigrees, compared to mRNA levels in fibroblasts from unaffected individuals. By contrast, mRNA levels encoding the a subunit of the G protein that inhibits adenylyl cyclase were not altered. Our findings suggest that several molecular mechanisms produce aG. deficiency in patients with pseudohypoparathyroidism type Ia and that major gene rearrangements or deletions are not a common cause for aG5 deficiency in pseudohypoparathyroidism type I.

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