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M C Keightley

Publications and source records attributed to M C Keightley.

9 recordsLinked to original sources
PubMed1998

Steroid receptor isoforms: exception or rule?

Since the first steroid receptor was cloned, it was quickly identified as one of many such receptors constituting a gene superfamily which has grown to include not only steroid receptors but also receptors for thyroid hormone, retinoic acid, 1,25-dihydroxyvitamin D3 as well as a number of less traditional ligands, including farnesoids and fatty acids. Interestingly, these receptors are far outnumbered by the 'orphan' receptors for which ligands are still being sought. The original cloning of nuclear receptors, although sometimes identifying more than one receptor form, led to the general premise that each ligand has its cognate receptor through which signal is transduced to the transcriptional machinery. Regulation of this process was found to occur at the level of receptor expression, ligand availability, and more recently, through post-translational modifications of the receptor and interaction of a variety of coactivators/corepressors with the receptor protein. The continuing identification of more than a single form for many of the receptors directed the attention of a number of investigators toward defining possible roles for these 'extras'. This review examines the different forms of nuclear receptor gene family members and how they may provide an additional level of regulation.

Animals↗
PubMed1998

Structural determinants of cortisol resistance in the guinea pig glucocorticoid receptor.

The guinea pig exhibits resistance to glucocorticoids in vivo which results from the guinea pig glucocorticoid receptor (GR) having a lower affinity for cortisol than the human GR. Cloning of the guinea pig GR has revealed that the amino acid sequence of the ligand-binding domain (LBD) differs from the human GR at 24 residues. The present study confirms that the decreased sensitivity and binding affinity of the guinea pig GR are conferred in vitro by the LBD. Further, the substitutions in the LBD do not confer altered relative steroid sensitivity or selectivity compared with the human GR. The altered sensitivity and binding of dexamethasone are confined to the first third of the LBD, which contains 5 nonconservative substitutions in a region that is otherwise highly conserved across several species of GR. These residues, either alone or in combination, were targeted for site-directed mutagenesis in both the human and guinea pig LBD. Trans-activation studies with these mutant GR failed to exclusively implicate or exclude any of the residues in the observed resistance. Rather, the changes, with 1 exception, caused a decrease in sensitivity, suggesting that critical intramolecular interactions involving at least 4 of these residues determine the correct conformation of this region. Recent molecular modeling of the GR LBD structure suggests that although the above region is not part of the core ligand-binding pocket, it is required to maintain the conformation of the binding pocket.

Amino Acid Sequence↗
PubMed1997

Determinants of specificity of transactivation by the mineralocorticoid or glucocorticoid receptor.

Glucocorticoids and mineralocorticoids have distinct in vivo roles despite close structural homology and similarities in vitro. Known mechanisms of specificity focus on factors extrinsic to the receptor; interactions that directly regulate the receptor to confer specificity are less well understood, particularly for the mineralocorticoid receptor (MR). To examine relative MR vs. glucocorticoid receptor (GR) function in a more physiological context, we compared transactivation by GR and MR in the standard experimental fibroblast CV-1 cell line, the renal epithelial LLC-PK1 line, and neuronal medullary raphe RN33B cells. Maximal transactivational activity mediated by MR, relative to that mediated by GR, is enhanced in both of these cell lines and is primarily conferred by an N-terminal-mediated enhancement of the MR response. In addition, the ligand concentration required for maximal transcriptional activity of the GR varies significantly between cell lines. This is independent of binding affinity or 11beta-hydroxysteroid dehydrogenase-mediated inactivation and may contribute to in vivo tissue-specific differences in responses to the GR. Although ligand binding affinity is clearly conferred by the LBD, receptor-specific variations between cell lines in transcriptional sensitivity to ligand appear, rather, to be associated with the N-terminus. These studies demonstrate that the specificity of the MR vs. the GR response may be mediated via unique cellular factors, as well as suggesting a novel means of expanding the cellular response to cortisol.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1996

The molecular basis of RU486 resistance in the Tammar Wallaby, Macropus eugenii.

RU486 acts as a potent anti-progestin in humans but does not antagonise progesterone action in the chicken or hamster reflecting a substitution in the ligand binding domain (LBD) of cysteine for glycine in both the chicken and the hamster progesterone receptor (PR), at the position corresponding to codon 722 of the human PR. The tammar wallaby, Macropus eugenii, is also resistant to the effects of RU486. Cloning of a partial cDNA of the PR in the tammar wallaby reveals a glycine to alanine substitution (gly 722 in the human PR), as well as a glutamine to histidine substitution two amino acids upstream of this alanine residue. Both the glycine and glutamine residues are substituted in all three resistant species. These substitutions are also found in the mineralocorticoid receptor, which also does not bind RU486, and suggest an important role for these residues in the formation of the 11-beta pocket of the receptor, which accommodates the bulky side-chains of 11-beta substituted steroids.

Amino Acid Sequence↗
PubMed1995

Human mineralocorticoid receptor genomic structure and identification of expressed isoforms.

Most of the known effects of aldosterone are mediated by the mineralocorticoid receptor, an intracellular receptor belonging to the steroid/thyroid hormone/retinoic acid receptor superfamily. We determined the genomic structure of the human MR (hMR) and identified 10 exons in the gene, including two exons (1 alpha and 1 beta) that encode different 5'-untranslated sequence. Expression of the two different hMR variants is under the control of two different promoters that contain no obvious TATA element, but multiple GC boxes. Our results indicate that hMR expression is regulated by alternative promoters perhaps in a tissue- or developmental-specific manner.

Bacteriophage M13↗
PubMed1995

Cortisol resistance and the guinea pig glucocorticoid receptor.

The guinea pig has been employed as a model to study the structure/function relationships of the glucocorticoid receptor (GR), and to determine the regions of the receptor important for binding hormone and antihormone. Guinea pigs have high levels of circulating cortisol and GR with a approximately 20-fold lower affinity for dexamethasone than mouse GR. Cloning and sequencing of guinea pig GR has identified 24 amino acid changes in the ligand-binding domain (LBD) compared to the human GR. By substituting the guinea pig GR LBD for the human LBD in a human GR expression vector we have shown in cotransfection studies that guinea pig GR LBD confers glucocorticoid resistance as observed in vivo. In initial studies guinea pig GR LBD appeared constitutively active; in subsequent studies to determine which of the 24 amino acid changes present in the guinea pig GR LBD conferred resistance, it became apparent that the guinea pig LBD (LBD delta), amplified by PCR for subcloning into the human GR expression vector, contained a single adenine deletion in the hinge region within ten bases of the PCR primer. This single base deletion resulted in a frameshift bringing a stop codon into frame one codon after the deletion. While this now clearly accounts for the observed constitutive activity, since it is known that C-terminally truncated steroid receptors exhibit constitutive activation such a truncation is more difficult to reconcile with the repeatedly demonstrable hormone dose-response curves obtained with this guinea pig GR LBD delta.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Unique sequences in the guinea pig glucocorticoid receptor induce constitutive transactivation and decrease steroid sensitivity.

Previous attempts to characterize the structural determinants required for binding of cortisol by the glucocorticoid receptor (GR) have proved difficult since almost all modifications of the ligand binding domain (LBD) of GRs either eliminate or greatly decrease steroid binding. The guinea pig, a New World hystricomorph with a phylogeny the subject of recent dispute, is corticoresistant due to a GR that has diminished affinity for dexamethasone. The guinea pig GR has been cloned, and sequencing has identified many unique amino acid substitutions in the LBD. Using a domain-swap approach, the cloned guinea pig GR LBD was substituted for the human GR LBD in a human GR expression vector. Dexamethasone response curves for these constructs show that the cortisol resistance observed in the guinea pig in vivo is conferred in vitro by the guinea pig GR LBD. In addition, the guinea pig GR LBD induces a high level of constitutive activity. This constitutive activity is not repressed by RU486 (1 microM) but is enhanced by the addition of 8-bromo-cAMP. One of the amino acid substitutions results in the loss of a cysteine, which in the human, rat, and mouse GR is the site of covalent attachment for dexamethasone-21-mesylate. This cysteine is replaced by a tryptophan residue in the guinea pig GR, the implications of which were examined by reciprocal mutation of the tryptophan to a cysteine in the guinea pig GR LBD, and the cysteine to a tryptophan in the human GR LBD.(ABSTRACT TRUNCATED AT 250 WORDS)

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1991

Molecular cloning and sequencing of a guinea-pig pro-opiomelanocortin cDNA.

The guinea-pig has high levels of circulating cortisol. Though adrenocorticotropin (ACTH) levels are similar to those in other mammals, guinea-pig ACTH has been reported to have a single amino-acid substitution which results in increased bioactivity of the peptide. Pro-opiomelanocortin (POMC) is the precursor for ACTH, gamma-melanocyte-stimulating hormone (gamma-MSH) and the endogenous opioid peptide beta-endorphin. Both to confirm this substitution in guinea-pig ACTH and to establish whether other non-conservative substitutions occur elsewhere in the precursor we cloned guinea-pig POMC. The guinea-pig alanine for proline substitution at position 24 of ACTH was confirmed. Potentially significant mutations were also identified in gamma-MSH and beta-endorphin. A similar pattern of POMC mRNA expression was obtained for guinea-pig and rat as determined by Northern analysis and in situ hybridization. Southern blot analysis indicated that guinea-pig POMC is a single-copy gene. Cloning and sequencing of guinea-pig POMC thus further demonstrate the divergence of the New World hystricomorph peptides from those in New World primates, and underscore the differences observed in other endocrine axes in the guinea-pig.

Adrenocorticotropic Hormone↗