Linked Life Data

10521440

Source:http://linkedlifedata.com/resource/pubmed/id/10521440

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
43
pubmed:dateCreated
1999-11-23
pubmed:abstractText
High affinity agonist binding to G protein-coupled receptors depends on the formation of a ternary complex between agonist, receptor, and G protein. This process is too slow to be accounted for by a simple diffusion-controlled mechanism. We have tested if the interaction between activated receptor and G protein is rate-limiting by fusing the coding sequence of the human A(1)-adenosine receptor to that of Galpha(i-1) (A(1)/Galpha(i-1)) and of Galpha(o) (A(1)/Galpha(o)). Fusion proteins of the expected molecular mass were detected following transfection of HEK293 cells. Ternary complex formation was monitored by determining the kinetics for binding of the high affinity agonist (-)-N(6)-3[(125)I](iodo-4-hydroxyphenylisopropyl)adenosine; these were similar in the wild-type receptor and the fusion proteins over the temperature range of 10 to 30 degrees C. Agonist dissociation may be limited by the stability of the ternary complex. This assumption was tested by creating fusion proteins in which the Cys(351) of Galpha(i-1) was replaced with glycine (A(1)/Galpha(i-1)C351G) or isoleucine (A(1)/Galpha(i-1)C351I) to lower the affinity of the receptor for the G protein. In these mutated fusion proteins, the dissociation rate of the ternary complex was accelerated; in contrast, the rate of the forward reaction was not affected. We therefore conclude that (i) receptor activation per se rather than its interaction with the G protein is rate-limiting in ternary complex formation; (ii) the stability of the ternary complex is determined by the dissociation rate of the G protein. These features provide for a kinetic proofreading mechanism that sustains the fidelity of receptor-G protein coupling.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Oct
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
22
pubmed:volume
274
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
30571-9
pubmed:dateRevised
2009-11-19
pubmed:meshHeading
pubmed:year
1999
pubmed:articleTitle
Kinetics of ternary complex formation with fusion proteins composed of the A(1)-adenosine receptor and G protein alpha-subunits.
pubmed:affiliation
Institute of Pharmacology, University of Vienna, Währinger Strasse 13a, A-1090 Vienna, Austria.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't