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J S Davidson

Publications and source records attributed to J S Davidson.

At least 19 recordsLinked to original sources
PubMed1998

Functional microdomains in G-protein-coupled receptors. The conserved arginine-cage motif in the gonadotropin-releasing hormone receptor.

An Arg present in the third transmembrane domain of all rhodopsin-like G-protein-coupled receptors is required for efficient signal transduction. Mutation of this Arg in the gonadotropin-releasing hormone receptor to Gln, His, or Lys abolished or severely impaired agonist-stimulated inositol phosphate generation, consistent with Arg having a role in receptor activation. To investigate the contribution of the surrounding structural domain in the actions of the conserved Arg, an integrated microdomain modeling and mutagenesis approach has been utilized. Two conserved residues that constrain the Arg side chain to a limited number of conformations have been identified. In the inactive wild-type receptor, the Arg side chain is proposed to form an ionic interaction with Asp3.49(138). Experimental results for the Asp3. 49(138) --> Asn mutant receptor show a modestly enhanced receptor efficiency, consistent with the hypothesis that weakening the Asp3. 49(138)-Arg3.50(139) interaction by protonation of the Asp or by the mutation to Asn favors activation. With activation, the Asp3. 49(138)-Arg3.50(139) ionic bond would break, and the unrestrained Arg would be prevented from orienting itself toward the water phase by a steric clash with Ile3.54(143). The mutation Ile3.54(143) --> Ala, which eliminates this clash in simulations, causes a marked reduction in measured receptor signaling efficiency, implying that solvation of Arg3.50(139) prevents it from functioning in the activation of the receptor. These data are consistent with residues Asp3.49(138) and Ile3.54(143) forming a structural motif, which helps position Arg in its appropriate inactive and active receptor conformations.

Amino Acid Sequence↗
PubMed1998

The role of antibiotics and timing of repair in flexor tendon injuries of the hand.

The use of antibiotics and the timing of repair in the management of flexor tendon injuries of the hand have never been specifically addressed in the literature. In a retrospective study, 140 patients treated for simple open flexor tendon injuries were evaluated for postoperative wound infection. Patients were divided into four groups on the basis of early or late repair and use of parenteral perioperative antibiotic therapy. No significant differences were found among groups with respect to the low incidence of infectious complications observed. There is no evidence that mandates the use of prophylactic antibiotics and immediate repair in these types of injuries.

Adult↗
PubMed1997

Irreversible activation of the gonadotropin-releasing hormone receptor by photoaffinity cross-linking: localization of attachment site to Cys residue in N-terminal segment.

Photoaffinity cross-linking with [azidobenzoyl-d-Lys6]GnRH leads to irreversible activation of the gonadotropin-releasing hormone (GnRH) receptor. In order to localize the cross-linking site, the disulfide bridge structure was initially probed by mutagenesis. A consistent pattern of changes in the ability of GnRH to stimulate signal transduction after Ser substitutions of extracellularly located Cys residues indicated that Cys14 in the N-terminal domain is connected to Cys200 in the second extracellular loop, while Cys196 in this loop is connected to the highly conserved Cys114 at the extracellular end of transmembrane helix 3. Protein chemical analysis of radioactive fragments of cross-linked GnRH receptor following deglycosylation and enzymatic digest with endoproteinase Glu-C and trypsin before and after introduction or elimination of potential protease cleavage sites indicated that 125I[azidobenzoyl-d-Lys6]GnRH cross-links to a segment comprising residues 12-18 of the N-terminal domain. The existence of the Cys114-Cys196 bridge was directly confirmed as a labeled fragment, including that Cys114 was resolvable only under reducing conditions. The observation that the cross-linked N-terminal enzymatic fragments had identical apparent size under non-reducing conditions shows that the cross-linking reaction disconnected the disulfide bridge between Cys14 and Cys200 and indicates that Cys14 is probably the residue involved in cross-linking of the ligand. It is concluded that covalent tethering of GnRH through a photoreactive side chain located at position 6 in the middle of the peptide leads to continued activation of the receptor presumably through covalent binding to Cys14 in the N-terminal domain of the receptor.

Amino Acid Sequence↗
PubMed1996

Desensitization of gonadotropin-releasing hormone action in alphaT3-1 cells due to uncoupling of inositol 1,4,5-trisphosphate generation and Ca2+ mobilization.

Gonadotropin-releasing hormone (GnRH) acts via a G-protein coupled receptor on gonadotropes to increase cytosolic Ca2+ and stimulate gonadotropin secretion. Sustained exposure causes desensitization of these effects, but the GnRH receptor has no C-terminal tail and does not undergo rapid (<5 min) desensitization. Nevertheless, pretreatment of alphaT3-1 cells with GnRH reduced the spike Ca2+ response to GnRH and decreased the GnRH effect on inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) by 30-50%. Ca2+-free medium with or without thapsigargin also decreased GnRH-stimulated Ins(1,4,5)P3 generation, implying that attenuation of the Ca2+ response underlies the Ins(1,4,5)P3 reduction rather than vice versa. Intracellular Ca2+ pool depletion cannot explain desensitization of the Ca2+ response because pool depletion and repletion were faster (half-times, <1 min) than the onset of and recovery from desensitization (half-times 10-20 min and 4-6 h). Moreover, 1-h GnRH pre-treatment attenuated the spike Ca2+ response to GnRH but not that to ionomycin, and brief GnRH exposure in Ca2+-free medium reduced the response to ionomycin more effectively in controls than in desensitized cells. GnRH pretreatment also attenuated the Ca2+ response to PACAP38. This novel form of desensitization does not reflect uncoupling of GnRH receptors from their immediate effector system but rather a reduced efficiency of mobilization by Ins(1,4,5)P3 of Ca2+ from an intact intracellular pool.

Calcium↗
PubMed1996

Asn102 of the gonadotropin-releasing hormone receptor is a critical determinant of potency for agonists containing C-terminal glycinamide.

We demonstrate a critical role for Asn102 of the human gonadotropin-releasing hormone (GnRH) receptor in the binding of GnRH. Mutation of Asn102, located at the top of the second transmembrane helix, to Ala resulted in a 225-fold loss of potency for GnRH. Eight GnRH analogs, all containing glycinamide C termini like GnRH, showed similar losses of potency between 95- and 750-fold for the [Ala102]GnRHR, compared with wild-type receptor. In contrast, four GnRH analogs that had ethylamide in place of the C-terminal glycinamide residue, showed much smaller decreases in potency between 2.4- and 11-fold. In comparisons of three agonist pairs, differing only at the C terminus, glycinamide derivatives showed an 11-20-fold greater loss of potency for the mutant receptor than their respective ethylamide derivatives. Thus Asn102 is a critical determinant of potency specifically for ligands with C-terminal glycinamide, while ligands with C-terminal ethylamide are less dependent on Asn102. These findings indicate a role for Asn102 in the docking of the glycinamide C terminus and are consistent with hydrogen bonding of the Asn102 side chain with the C-terminal amide moiety. Taken with previous data, they suggest a region of the GnRH receptor formed by the top of helices 2 and 7 as a binding pocket for the C-terminal part of the ligand.

Amino Acid Sequence↗
PubMed1996

Gluteus maximus musculocutaneous flap for the treatment of recalcitrant pilonidal disease.

Although conservative surgery should be the initial approach to pilonidal disease, there remains a select group of patients with recalcitrant disease who fail repeated interventions and for whom a more aggressive approach is justified. Use of a large gluteus maximus musculocutaneous flap to facilitate wide excision, to allow for primary closure with well-vascularized tissue, and to alter the anatomy of the gluteal cleft has been used in 5 patients with chronic recurrent recalcitrant disease. The use of this large muscle unit in these otherwise healthy adults has achieved control over their pilonidal disease with acceptable morbidity and no demonstrable functional sequelae.

Adult↗
PubMed1996

Ca2+ entry in gonadotrophs and alpha T3-1 cells: does store-dependent Ca2+ influx mediate gonadotrophin-releasing hormone action?

In pituitary gonadotrophs GnRH causes biphasic (spike and plateau) increases in cytosolic Ca2+ ([Ca2+]i) and gonadotrophin release. The spike phases reflect mobilization of stored Ca2+ and the plateau responses are attributed, in part, to Ca2+ influx via voltage-sensitive Ca2+ channels. In recent years, store-dependent Ca2+ influx (SDCI), in which depletion of the intracellular inositol 1,4,5-trisphosphate-mobilizable pool stimulates Ca2+ influx, has emerged as a major form of Ca2+ entry activated by phosphoinositidase C-coupled receptors in non-excitable cells. More recent evidence also indicates a role for SDCI in excitable cells. We have used dynamic video imaging of [Ca2+]i in alpha T3-1 cells (a gonadotroph-derived cell line) and manipulation of the filling state of the GnRH-mobilizable Ca2+ pool to test the possible role of SDCI in GnRH action. In Ca(2+)-containing medium, GnRH caused a biphasic increase in [Ca2+]i whereas in Ca(2+)-free medium only a transient increase occurred. The response to a second stimulation with GnRH in Ca(2+)-free medium was reduced by > 95% (demonstrating that Ca2+ pool depletion had occurred) and was recovered after brief exposure to Ca(2+)-containing medium (which enables refilling of the pool). Ionomycin (a Ca2+ ionophore) and thapsigargin (which inhibits the Ca(2+)-sequestering ATPase of the endoplasmic reticulum) also transiently increased [Ca2+]i in Ca(2+)-free medium and depleted the GnRH-mobilizable pool as indicated by greatly reduced subsequent responses to GnRH. Pool depletion also occurs on stimulation with GnRH in Ca(2+)-containing medium because addition of ionomycin and Ca(2+)-free medium during the plateau phase of the GnRH response caused only a reduction in [Ca2+]i rather than the transient increase seen without GnRH. To deplete intracellular Ca2+ pools, cells were pretreated in Ca(2+)-free medium with thapsigargin or GnRH and then, after extensive washing, returned to Ca(2+)-containing medium. Pretreatment with thapsigargin augmented the increase in [Ca2+]i seen on return to Ca(2+)-containing medium (to two- to threefold higher than that seen in control cells) indicating the activation of SDCI, whereas pool depletion by GnRH pretreatment had no such effect. To ensure maintained pool depletion after Ca2+ re-addition, similar studies were performed in which the thapsigargin and GnRH treatments were not washed off, but were retained through the period of return to Ca(2+)-containing medium. Return of GnRH-treated cells to Ca(2+)-containing medium caused an increase in [Ca2+]i which was inhibited by nicardipine, whereas the increase seen on return of thapsigargin-treated cells to Ca(2+)-containing medium was not reduced by nicardipine. The quench of fura-2 fluorescence by MnCl2 (used as a reporter of Ca2+ influx) was increased by GnRH and thapsigargin, indicating that both stimulate Ca2+ influx via Mn2+ permeant channels. The GnRH effect was abolished by nicardipine whereas that of thapsigargin was not. Finally, depletion of intracellular Ca2+ pools by pretreatment of superfused rat pituitary cells with GnRH or thapsigargin in Ca(2+)-free medium did not enhance LH release on return to Ca(2+)-containing medium. The results indicate that (a) thapsigargin stimulates SDCI in alpha T3-1 cells via nicardipine-insensitive Ca2+ channels, (b) in spite of the fact that GnRH depletes the hormone-mobilizable Ca2+ pool, it fails to stimulate SDCI, (c) GnRH stimulates Ca2+ entry predominantly via nicardipine-sensitive channels, a route not activated by SDCI and (d) in rat gonadotrophs, GnRH-stimulated LH release is not mediated by SDCI.

Animals↗
PubMed1995

Recombinant protein sequences can trigger methylation of N-terminal amino acids in Escherichia coli.

Recombinant human hemoglobin rHb1.1 has been genetically engineered with the replacement of the wild-type valine residues at all N-termini with methionine, an Asn 108 Lys substitution on the beta globins, and a fusion of the two alpha globins with a glycine linker. When rHb1.1 was expressed in Escherichia coli, methylation of the N-terminal methionine of the alpha globin was discovered. Another mutant has been engineered with the alpha globin gene coding for N-terminal methionine followed by an insertion of alanine. Characterization of expressed hemoglobin from this variant revealed a methylated N-terminal alanine that occurred through two posttranslational events: initial excision of the N-terminal methionine, followed by methylation of alanine as the newly generated N-terminus. No methylation was observed for variants expressed with wild-type valine at the N-terminus of the alpha globin. The methylation of N-terminal amino acids was attributed to a specific protein sequence that can trigger methylation of proteins expressed in E. coli. Here we demonstrate that proline at position 4 in the protein sequence of alpha globin seems an essential part of that signaling. Although N-terminal methylation has been observed previously for native E. coli proteins with similar N-terminal sequences, methylation of the recombinant globins has allowed further delineation of the recognition sequence, and indicates that methylation of heterologous proteins can occur in E. coli.

Alanine↗
PubMed1995

Identification of N-glycosylation sites in the gonadotropin-releasing hormone receptor: role in receptor expression but not ligand binding.

The asparagine residues of the three N-glycosylation consensus sequences in the mouse gonadotropin-releasing hormone receptor were mutated to determine which residues were glycosylated and the function of glycosylation. Photoaffinity labelled Gln4 and Gln18 receptor mutants exhibited lower apparent molecular weight on SDS polyacrylamide gel electrophoresis, while the Gln102 receptor showed wildtype mobility. This indicates that the receptor is glycosylated at Asn4 and Asn18 but not at Asn102. Binding affinities of all the mutant receptors were normal, indicating that carbohydrate moieties are not involved in ligand binding interactions. However, expression of the Gln4 and Gln18 receptors were substantially decreased, indicating a role for glycosylation in receptor expression or stability. All the glycosylation site mutants were capable of normal signal transduction, as indicated by their ability to stimulate inositol phosphate production.

Animals↗
PubMed1994

Glutamate 301 of the mouse gonadotropin-releasing hormone receptor confers specificity for arginine 8 of mammalian gonadotropin-releasing hormone.

The Arg residue at position 8 of mammalian GnRH is necessary for high affinity binding to mammalian GnRH receptors. This requirement has been postulated to derive from an electrostatic interaction of Arg8 with a negatively charged receptor residue. In order to identify such a residue, 8 conserved acidic residues of the mouse GnRH receptor were mutated to isosteric Asn or Gln. Mutant receptors were tested for decreased preference for Arg8-containing ligands by ligand binding and inositol phosphate production. One of the mutants, in which the Glu301 residue was mutated to Gln, exhibited a 56-fold decrease in apparent affinity for mammalian GnRH. The mutant receptor also exhibited decreased affinity for [Lys8]GnRH, but its affinity for [Gln8]GnRH was unchanged compared with the wild type receptor. The apparent affinity of the mutant receptor for the acidic analogue, [Glu8]GnRH, was increased more than 10-fold. The mutant receptor did not, therefore, distinguish mammalian GnRH from analogues with amino acid substitutions at position 8 as effectively as the wild type receptor. This loss of discrimination was specific for the residue at position 8, because the mutant receptor did distinguish mammalian GnRH from analogues with favorable substitutions at positions 5, 6, and 7. These findings show that Glu301 of the GnRH receptor plays a role in receptor recognition of Arg8 in the ligand and are consistent with an electrostatic interaction between these 2 residues.

Amino Acid Sequence↗
PubMed1994

Absence of rapid desensitization of the mouse gonadotropin-releasing hormone receptor.

Desensitization of gonadotropin release by the pituitary gland in response to gonadotropin-releasing hormone (GnRH) agonists has clinical applications in the treatment of gonadal-hormone-dependent disorders. We therefore investigated possible desensitization of inositol phosphate (IP) responses of GNRH receptors. No short-term homologous desensitization of the IP response to GnRH was observed in either alpha T3 gonadotrope cells line or GH3 cells transfected with GnRH receptor cDNA. The absence of homologous desensitization is unusual among G-protein-coupled receptors, and may be due to the absence of a C-terminal cytoplasmic tail, a unique feature of the GnRH receptor. Several potential protein kinase C phosphorylation sites which might mediate heterologous desensitization are present on the GnRH receptor. In both alpha T3 cells and GnRH-receptor-transfected Cos-1 cells, activation of protein kinase C by pretreatment with phorbol ester caused a 35-53% decrease in the IP response to GnRH. However, phorbol ester also inhibited guanosine 5'-[gamma-thio]triphosphate-stimulated IP production in permeabilized Cos-1 cells, suggesting that this inhibition is mediated at a post-receptor site.

Animals↗
PubMed1994

A reciprocal mutation supports helix 2 and helix 7 proximity in the gonadotropin-releasing hormone receptor.

Activation of the pituitary gonadotropin-releasing hormone receptor, a member of the seven-transmembrane G protein-coupled receptor (GPCR) family, triggers a cascade of events leading to gonadotropin release and stimulation of the reproductive system. An unusual feature of this receptor, observed in mice, rats, and humans, is the presence of Asn87 in the second putative transmembrane helix at the location of a highly conserved aspartate in the GPCR family and of Asp318 in the putative seventh transmembrane helix where nearly all other GPCRs have asparagine. The possibility that these residues interact was suggested by this reciprocal pattern and by a three-dimensional model of the gonadotropin-releasing hormone receptor and was investigated by site-directed mutagenesis. Replacing Asn87 in the second transmembrane domain by aspartate eliminated detectable ligand binding. A second mutation, generating the double-mutant receptor Asp87Asn318, recreated the arrangement found in other GPCRs and re-established high affinity agonist and antagonist binding. The restoration of binding by a reciprocal mutation indicates that these two specific residues in helices 2 and 7 are adjacent in space and provides an empirical basis to refine the model of the transmembrane helix bundle of the receptor.

Amino Acid Sequence↗
PubMed1993

Comparative sequence analysis and functional characterization of the cloned sheep gonadotropin-releasing hormone receptor reveal differences in primary structure and ligand specificity among mammalian receptors.

The cloned sheep gonadotropin-releasing hormone (GnRH) receptor was analysed for sequence homology/differences among mammalian receptors and its pharmacology characterized in COS-1 cells. Transmembrane domains TM2, TM3, TM5, TM6 and TM7, and extracellular loop 1 are most highly conserved (> 90%) in this G-protein coupled receptor. The Kd of the sheep receptor in binding assays (4.9 nM) was similar to the human and rat receptors, but lower than the mouse receptor. The rank order of potency of a series of GnRH analogues for binding and inositol phosphate stimulation in transfected COS-1 cells was identical to that of the receptor characterized in sheep pituitary gonadotropes. Northern blot analysis identified four transcripts sized 5.4 kb, 3.6 kb, 2.3 kb and 1.3 kb in sheep pituitaries which were upregulated by castration.

Amino Acid Sequence↗
PubMed1993

Inhibition of pituitary hormone exocytosis by a synthetic peptide related to the rab effector domain.

GTP-binding proteins of the rab family are believed to function at several steps in intracellular vesicular transport. We examined the effects of a rab-related peptide in permeabilized pituitary cells, in which exocytosis can be triggered by distinct Ca(2+)-dependent or Ca(2+)-independent pathways. We report that a synthetic peptide of 18 amino acids related to the rab effector domain, rab3AL (30-47) inhibited luteinizing hormone (LH) and growth hormone (GH) exocytosis triggered by either pathway. Ca(2+)-stimulated LH and GH release were inhibited by more than 80% and 50%, respectively, by 100 microM peptide. The peptide (100 microM) also inhibited LH and GH exocytosis stimulated by phorbol myristate acetate plus cAMP by more than 45% and 80%, respectively. The effect was sequence-specific since a second peptide, lacking the first 3 amino acids but otherwise identical failed to inhibit exocytosis. These results suggest that a protein of the rab family is involved in regulated pituitary hormone exocytosis, and they identify 3 amino acids of the putative rab effector domain which may be functionally important in exocytosis.

Amino Acid Sequence↗
PubMed1993

Cloning and characterization of the human GnRH receptor.

A cDNA encoding the human GnRH receptor (GnRHR) has been cloned and functionally expressed in both Xenopus oocytes and COS-1 cells. The 2160 bp cDNA encodes a 328 amino acid protein with a predicted amino acid sequence that is 90% identical to that of the mouse GnRHR (Tsutsumi et al. (1992) Mol. Endocrinol. 6, 1163-1169). Injection of synthetic RNA transcript into oocytes led to the development of a depolarizing response to agonists when assayed by voltage-clamp electrophysiology. Consistent with the expression of a mammalian GnRHR, the response was blocked by GnRH antagonists. Following expression of the human GnRHR in COS-1 cells, agonists and an antagonist displaced [125I]GnRH agonist from membrane isolates with nanomolar range dissociation constants similar to those described for displacement from human pituitary membranes. Transfected COS-1 cells manifested a GnRH-stimulated increase in phosphoinositol turnover, with an EC50 of approximately 3 nM, which was inhibited by GnRH antagonists. Northern blot analysis revealed a single band of approximately 4.7 kb expressed in human pituitary which was not detected in testis. The predicted structure of the human GnRHR is similar to that previously reported for the mouse receptor. Although the mammalian GnRHR is a seven transmembrane domain receptor, it differs from other G-protein coupled receptors in several respects, most notably the lack of a cytoplasmic C-terminal domain. The present study demonstrates that the cDNA isolated encodes the human GnRHR and suggests that several unique features conserved among mammalian GnRHRs may be essential for receptor function and/or regulatory control.

Amino Acid Sequence↗
PubMed1993

Stimulation by Mn2+ and inhibition by Cd2+, Zn2+, Ni2+, and Co2+ ions of luteinizing hormone exocytosis at an intracellular site.

Direct intracellular effects of the divalent cations Mn2+, Cd2+, Co2+, Ni2+, and Zn2+ on pituitary hormone exocytosis in a permeabilized cell system have not previously been investigated. We examined the effects of these ions on LH exocytosis in primary sheep pituitary cells permeabilized with Staphylococcal alpha-toxin. Mn2+ ions strongly stimulated LH release from permeabilized, but not intact, cells. Mn(2+)-stimulated LH release was ATP-dependent and sensitive to N-ethyl maleimide, indicating that it represents true exocytosis. Hormone release triggered by Ca2+ was inhibited by Cd2+, Zn2+, Co2+, and Ni2+ ions. Half-maximal inhibition of Ca(2+)-stimulated LH release was observed with 10 microM Cd2+, 30 microM Zn2+, 0.3 mM Co2+, and 1 mM Ni2+. With the same order of potency these ions inhibited LH release stimulated by Ba2+, Mn2+, or phorbol ester plus cAMP, suggesting that they inhibit exocytosis at an intracellular site common to all these stimuli.

Adenosine Triphosphate↗
PubMed1992

Sequence and methylation in the beta/A4 region of the rabbit amyloid precursor protein gene.

Alzheimer's disease is characterized by the accumulation of the beta/A4 fragment of the amyloid precursor protein in the hippocampal regions of the brain. We report here the isolation of genomic clones carrying exons 15, 16 and 17 of the beta/A4 coding region of the rabbit amyloid precursor protein gene. The complete sequence of these exons predicts that all three peptides are identical to their human counterparts. An unexpectedly high concentration of CpG dinucleotides seen in exon 15 were conserved and continued into the intron 15 region. MspI/HpaII southern blot analysis revealed the presence of a number of methylated CpG dinucleotides in the cloned region of the gene. These data suggest that the rabbit amyloid precursor protein gene could provide a new and useful model for the study of this important gene.

Amino Acid Sequence↗