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Biomedical subjects

A Shenker

Publications and source records attributed to A Shenker.

35 records · Page 2Linked to original sources
PubMed1994

An activating Gs alpha mutation is present in fibrous dysplasia of bone in the McCune-Albright syndrome.

McCune-Albright syndrome (MAS) is a sporadic disease characterized by polyostotic fibrous dysplasia, café-au-lait spots, and multiple endocrinopathies. The etiology of fibrous dysplasia is unknown. Activating mutations of codon 201 in the gene encoding the alpha-subunit of Gs, the G-protein that stimulates adenylyl cyclase, have been found in all affected MAS tissues that have been studied. Initial attempts to amplify DNA from decalcified paraffin-embedded bone specimens from MAS patients were unsuccessful. Therefore, we analyzed DNA from frozen surgical bone specimens from five MAS patients using polymerase chain reaction and allele-specific oligonucleotide hybridization. Most of the cells in four specimens of dysplastic bone contained a heterozygous mutation encoding substitution of Arg201 of Gs alpha with His, but the mutation was barely detectable in peripheral blood specimens from the patients. Only a small amount of mutant allele was detected in a specimen of normal cortical bone from the fifth patient, although this patient had a high proportion of mutation in other, affected tissues. The mosaic distribution of mutant alleles is consistent with an embryological somatic cell mutation of the Gs alpha gene in MAS. The presence of an activating mutation of Gs alpha in osteoblastic progenitor cells may cause them to exhibit increased proliferation and abnormal differentiation, thereby producing the lesions of fibrous dysplasia.

Adenylyl Cyclases↗
PubMed1993

A constitutively activating mutation of the luteinizing hormone receptor in familial male precocious puberty.

Familial male precocious puberty (FMPP) is a gonadotropin-independent disorder that is inherited in an autosomal dominant, male-limited pattern. Affected males generally exhibit signs of puberty by age 4. Testosterone production and Leydig cell hyperplasia occur in the context of prepubertal levels of luteinizing hormone (LH). The LH receptor is a member of the family of G-protein-coupled receptors, and we hypothesized that FMPP might be due to a mutant receptor that is activated in the presence of little or no agonist. A single A-->G base change that results in substitution of glycine for aspartate at position 578 in the sixth transmembrane helix of the LH receptor was found in affected individuals from eight different families. Linkage of the mutation to FMPP was supported by restriction-digest analysis. COS-7 cells expressing the mutant LH receptor exhibited markedly increased cyclic AMP production in the absence of agonist, suggesting that autonomous Leydig cell activity in FMPP is caused by a constitutively activated LH receptor.

Adenine↗
PubMed1993

G protein mutations in human disease.

The heterotrimeric G proteins couple cell-surface receptors for extracellular signals to intracellular effectors that generate second messengers. Abnormal G protein signalling, resulting from posttranslational modifications by bacterial toxins, altered gene expression, or gene mutations, may lead to diverse biological consequences. Mutations within G protein alpha subunit genes that lead to either constitutive activation or loss of function have been identified. Such G protein mutations play a role in the pathogenesis of several human diseases, including sporadic endocrine tumors, McCune-Albright syndrome, and Albright hereditary osteodystrophy.

Animals↗
PubMed1993

Severe endocrine and nonendocrine manifestations of the McCune-Albright syndrome associated with activating mutations of stimulatory G protein GS.

McCune-Albright syndrome (MCAS) is a sporadic disease classically including polyostotic fibrous dysplasia, café au lait spots, sexual precocity, and other hyperfunctional endocrinopathies. An activating missense mutation in the gene for the alpha subunit of GS, the G protein that stimulates cyclic adenosine monophosphate formation, has been reported to be present in these patients. The mutation is found in variable abundance in different affected endocrine and nonendocrine tissues, consistent with the mosaic distribution of abnormal cells generated by a somatic cell mutation early in embryogenesis. We describe three patients with MCAS who had profound endocrine and nonendocrine disease and who died in childhood. Two of the patients were severely ill neonates whose complex symptoms did not immediately suggest MCAS. A mutation of residue Arg201 of GS alpha was found in affected tissues from all three children. A review of the literature and unpublished case histories emphasizes the existence of other patients with severe and unusual clinical manifestations. We conclude that the manifestations of MCAS are more extensive than is generally appreciated, and may include hepatobiliary disease, cardiac disease, other nonendocrine abnormalities, and sudden or premature death.

Adolescent↗
PubMed1993

Substitutions of different regions of the third cytoplasmic loop of the thyrotropin (TSH) receptor have selective effects on constitutive, TSH-, and TSH receptor autoantibody-stimulated phosphoinositide and 3',5'-cyclic adenosine monophosphate signal generation.

TSH and immunoglobulin G (IgG) preparations from patients with Graves' disease increase inositol phosphate as well as cAMP formation in Cos-7 cells transfected with rat TSH receptor (TSHR) cDNA. In a previous report, we mutated alanine 623 of the third cytoplasmic loop (residues 605-625) of the TSHR and showed it was critical for TSH and Graves' IgG initiation of phosphatidylinositol bisphosphate (PIP2) but not cAMP signaling. In this report, we substituted residues in the third loop of the TSHR with sequences from the N- and C-termini of the third loop of the alpha 1- and beta 2-adrenergic receptors (ARs), which computer analysis has identified as homologous to those in the TSHR. Alanine 623 is conserved in most ARs as well as in glycoprotein hormone receptors; there is, therefore, no change in alanine 623. After transfection of the mutant TSHR cDNAs into Cos-7 cells, we show that the mutant proteins are normally synthesized, processed, and incorporated into the membrane bilayer by Western blotting with a specific receptor antibody. We also show that the dissociation constant for TSH binding in all mutants is the same or lower than wild type TSHR. We then evaluated the ability of TSH or Graves' IgG to increase PIP2 and cAMP signals in each transfectant. Mutants A622 and B621 replace, respectively, residues 622-625 and 621-625 of the TSHR with alpha 1- and beta 2-AR residues from the C-terminus of the third cytoplasmic loop; mutants A607 and B605 replace, respectively, TSHR residues 607-609 and 605-609 with N-terminus residues from alpha 1- and beta 2-AR. All four mutants, like the alanine 623 mutant, result in transfected cells which lose TSH and Graves' IgG initiation of PIP2 but not cAMP signalling. Like the alanine 623 mutation to glutamic acid, the A607, B605, A622, and B621 mutants also result in decreased basal cAMP, but not inositol phosphate levels, relative to wild type receptor. In contrast to these results, mutants A610, B610, A617, and B617, which replace residues 610-613 or 617-620 of the TSHR with corresponding residues of the alpha 1- and beta 2-AR, retain TSH and Graves' IgG responsiveness in both inositol phosphate and cAMP assays. Mutation of residues 610-613, in fact, potentiates TSH-increased inositol phosphate production, despite having no effect on TSH-increased cAMP production.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Mutation of alanine 623 in the third cytoplasmic loop of the rat thyrotropin (TSH) receptor results in a loss in the phosphoinositide but not cAMP signal induced by TSH and receptor autoantibodies.

Thyrotropin (TSH) and IgG preparations from patients with Graves' disease increase inositol phosphate as well as cAMP formation in Cos-7 cells transfected with rat TSH receptor cDNA. Mutation of alanine 623 in the carboxyl end of the third cytoplasmic loop of the TSH receptor, to lysine or glutamic acid, results in the loss of TSH- and Graves' IgG-stimulated inositol phosphate formation but not in stimulated cAMP formation. There is no effect of the mutations on basal or P2-purinergic receptor-mediated inositol phosphate formation. The mutations do not affect transfection efficiency or the synthesis, processing, or membrane integration of the receptor, as evidenced by the unchanged amount and composition of the TSH receptor forms on Western blots of membranes from transfected cells. The mutations increase the affinity of the TSH receptor for [125I]TSH and decrease Bmax; however, cells with an equivalently decreased Bmax as a result of transfection with lower levels of wild type receptor do not lose either TSH-induced inositol phosphate formation or cAMP signaling activity. Thus, in addition to discriminating between ligand-induced phosphatidylinositol bisphosphate and cAMP signals, the mutation appears to cause an altered receptor conformation which affects ligand binding to its large extracellular domain.

Alanine↗
PubMed1991

Activating mutations of the stimulatory G protein in the McCune-Albright syndrome.

BACKGROUND: The McCune-Albright syndrome is a sporadic disease characterized by polyostotic fibrous dysplasia, café au lait spots, sexual precocity, and hyperfunction of multiple endocrine glands. These manifestations may be explained by a somatic mutation in affected tissues that results in activation of the signal-transduction pathway generating cyclic AMP (cAMP). We analyzed DNA from tissues of patients with the McCune-Albright syndrome for the presence of activating mutations of the gene for the alpha subunit of the G protein (Gs alpha) that stimulates cAMP formation. METHODS: Genomic DNA fragments encompassing regions (exons 8 and 9) previously found to contain activating missense mutations of the Gs alpha gene (gsp mutations) in sporadically occurring pituitary tumors were amplified in tissues from four patients with the McCune-Albright syndrome by the polymerase chain reaction. The amplified DNA was analyzed for mutations by denaturing gradient gel electrophoresis and allele-specific oligonucleotide hybridization. RESULTS: We detected one of two activating mutations within exon 8 of the Gs alpha gene in tissues from all four patients, including affected endocrine organs (gonads, adrenal glands, thyroid, and pituitary) and tissues not classically involved in the McCune-Albright syndrome. In two of the patients, histidine was substituted for arginine at position 201 of Gs alpha, and in the other two patients cysteine was substituted for the same arginine residue. In each patient the proportion of cells affected varied from tissue to tissue. In two endocrine organs, the highest proportion of mutant alleles was found in regions of abnormal cell proliferation. CONCLUSIONS: Mutations within exon 8 of the Gs alpha gene that result in increased activity of the Gs protein and increased cAMP formation are present in various tissues of patients with the McCune-Albright syndrome. Somatic mutation of this gene early in embryogenesis could result in the mosaic population of normal and mutant-bearing tissues that may underlie the clinical manifestations of this disease.

Adolescent↗
PubMed1991

The G protein coupled to the thromboxane A2 receptor in human platelets is a member of the novel Gq family.

The thromboxane A2 (TXA2) receptor in human platelets is coupled to a pertussis toxin-insensitive G protein whose identity has remained unknown. Candidates for this role include the atypical G protein known as Gz and members of a recently discovered G protein family known as Gq. Because of the proven utility of antibodies directed against the C terminus of G protein alpha subunits as functional probes, we prepared an antibody against a synthetic decapeptide corresponding to the C-terminal sequence shared by alpha 11 and alpha q, two members of the new family. This antibody (QL) does not recognize known alpha subunits but selectively binds to a 42-kDa protein in a variety of tissues, including human platelet membranes. QL and two other C-terminal antibodies, QN and AS, known to recognize alpha z and alpha i2, respectively, were tested for their ability to block agonist-stimulated GTPase activity in human platelet membranes. Pretreatment of platelet membranes with AS has previously been shown to interfere with alpha 2 adrenergic receptor-mediated inhibition of adenylylcyclase. As expected, only AS antibody produced inhibition of alpha 2 receptor-stimulated GTPase. Pretreatment of membranes with QL, but not QN or AS, caused marked inhibition of TXA2 receptor-stimulated GTPase. This identifies the G protein coupled to human platelet TXA2 receptors as a member of the novel Gq family.

Amino Acid Sequence↗
PubMed1988

An unusual cause of right lower quadrant pain in a prepubertal child.

The clinical course of a five-year-old girl with right lower abdominal pain is presented. The differential diagnosis of right lower quadrant abdominal pain in a prepubertal child and a discussion of this child's diagnosis, ovarian torsion, are provided. The need always to consider gynecologic disease in any female patient presenting with a history of colicky lower abdominal pain or an acute abdomen is emphasized.

Abdomen, Acute↗
PubMed1987

Pharmacological characterization of two 5-hydroxytryptamine receptors coupled to adenylate cyclase in guinea pig hippocampal membranes.

Two 5-hydroxytryptamine (5-HT) receptors mediate stimulation of adenylate cyclase activity in membranes of adult guinea pig hippocampus. The two receptors were characterized with agonists and antagonists and with the aid of computerized curve-fitting procedures. Each receptor mediates about 50% of the maximal response to 5-HT. 5-HT is about 10-fold more potent in eliciting response through one cyclase-linked receptor (RH) than the other (RL). The concentrations of 5-HT that elicit half-maximal response through RH and RL are 43 +/- 6 nM and 414 +/- 53 nM, respectively. 5-Methoxytryptamine (5-MeOT) and 5-HT are approximately equipotent at each receptor. The agonists tryptamine and bufotenine are less potent than 5-HT at both receptors, and each is about 50-fold selective for RH. The two receptors are best discriminated by the agonists 5-carboxamidotryptamine (5-CONH2-T) and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), both of which are selective for RH. 5-CONH2-T is about 7-fold more potent than 5-HT at RH. The rank order of agonist potencies at RH (5-CONH2-T greater than 8-OH-DPAT = 5-HT = 5-MeOT greater than bufotenine greater than tryptamine) differs from that at RL (5-HT = 5-MeOT greater than bufotenine greater than tryptamine = 5-CONH2-T greater than 8-OH-DPAT). Spiperone acts as a simple competitive antagonist at RH, with a dissociation constant of 20 nM, but it is at least 100-fold less potent as an antagonist at RL. The relatively low affinities of the selective 5-HT antagonists ketanserin and MDL 72222 for RH and RL indicate that neither receptor may be classified as the 5-HT2 or as the 5-HT3 (i.e., peripheral neuronal) type. The characteristics of RH suggest that it is a functional correlate of the 5-HT1A-binding site in brain. RL appears not to correspond to a known 5-HT-binding site, but it may be homologous to receptors that mediate 5-HT-stimulated adenylate cyclase activity in other systems such as infant rat colliculi. RH and RL may also mediate stimulation of adenylate cyclase activity by 5-HT in hippocampal membranes of adult rat.

5-Methoxytryptamine↗
PubMed1983

Enhanced serotonin-stimulated adenylate cyclase activity in membranes from adult guinea pig hippocampus.

We have developed an assay for serotonin (5-HT) stimulation of adenylate cyclase activity in membranes from adult guinea pig hippocampus. The response to 5-HT is concentration-dependent, with an EC50 of 0.01 microM, a shallow slope, and mean maximal stimulation of 90% over basal activity. The response to 5-HT is GTP-dependent and additive to the maximal stimulation by histamine. Micromolar concentrations of the known 5-HT receptor agonists, tryptamine and 5-methoxytryptamine, also stimulate cAMP production in this system, and their effect is not additive to that elicited by a maximal concentration of 5-HT. These results are consistent with the hypothesis that the response to 5-HT is elicited through a distinct receptor coupled to adenylate cyclase; the magnitude and the reproducibility of the 5-HT response in this system should make it useful for receptor classification. To examine the effect of prior exposure to endogenous 5-HT on the responsiveness of the system, we assayed 5-HT stimulation of enzyme activity in membranes prepared from animals 25-27 hrs after treatment with a single injection of reserpine (5 mg/kg, i.p.). The mean maximal stimulation of adenylate cyclase by 5-HT was increased to 150% over basal activity with no effect on the EC50 or slope of the 5-HT concentration-response curve. Reserpine pretreatment did not affect basal activity or histamine-stimulated adenylate cyclase activity. These results are discussed in the context of a hypothesis that endogenous 5-HT normally exerts a desensitizing effect on its receptors in situ.

5-Methoxytryptamine↗