Linked Life Data

9202220

Source:http://linkedlifedata.com/resource/pubmed/id/9202220

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
7
pubmed:dateCreated
1997-7-24
pubmed:abstractText
In this study, site-directed mutagenesis and biochemical strategies have been used to establish whether disulfide bonding between extracellular Cys residues contributes to the structural integrity of the GnRH receptor (GnRH-R) and, if so, to delineate the nature of the bonding patterns involved. The majority of G protein-coupled receptors (GPCRs) contain a pair of conserved Cys residues in the first and second extracellular domains, and these residues have been shown to form a receptor stabilizing disulfide bridge structure. However, many GPCRs contain other nonconserved Cys residues, and in some GPCRs these have also been shown to contribute to receptor integrity and stability. The rat GnRH-R contains four extracellular Cys residues. Two are conserved throughout the GPCR superfamily and lie at positions Cys114 and Cys195 in the first and second extracellular loops, respectively. The other two Cys residues occupy nonconserved positions at Cys14 in the amino terminus and Cys199 in the second extracellular loop. To assess the role of extracellular Cys residues in disulfide bonding interactions, each of these residues were mutated to Ala, expressed in COS-1 cells, and ligand binding and second messenger properties ascertained. To monitor levels of wild-type (WT) and mutant receptor cell surface expression, a hemagglutinin (HA) epitope tag was incorporated into the receptor constructs (GnRH-R WT, Cys14Ala, Cys114Ala, Cys195Ala, and Cys199Ala). Cys199Ala mutant maintained levels of receptor binding and second messenger production comparable with the WT GnRH-R, whereas mutant Cys14Ala exhibited some ligand binding and functional receptor activity, albeit at a reduced level. Mutations Cys114Ala and Cys195Ala showed no functional responses despite displaying levels of cell surface expression similar to the WT receptor. Specific binding of the WT and mutant receptors Cys14Ala and Cys199Ala was inhibited in the presence of the disulfide bond reducing agent, DTT, implying that disulfide bonds are formed and can be reduced in these mutant receptors. This study demonstrates that GnRH-R residues Cys114 and Cys195 have a disulfide bonding interaction role essential for the maintenance of receptor function. In contrast, Cys14 and Cys199 are not involved in disulfide bonding that is required for ligand binding or second messenger production.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
AIM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jul
pubmed:issn
0013-7227
pubmed:author
pubmed:issnType
Print
pubmed:volume
138
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
2800-6
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
1997
pubmed:articleTitle
An intramolecular disulfide bond between conserved extracellular cysteines in the gonadotropin-releasing hormone receptor is essential for binding and activation.
pubmed:affiliation
Medical Research Council Reproductive Biology Unit, Centre for Reproductive Biology, Edinburgh, United Kingdom.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't