| Autor: |
Ward, Stuart D.C., Hamdan, Fadi F., Bloodworth, Lanh M., Wess, Jürgen |
| Zdroj: |
Journal of Biological Chemistry; January 2002, Vol. 277 Issue: 3 p2247-2257, 11p |
| Abstrakt: |
The structural changes involved in ligand-dependent activation of G protein-coupled receptors are not well understood at present. To address this issue, we developed an in situdisulfide cross-linking strategy using the rat M3muscarinic receptor, a prototypical Gq-coupled receptor, as a model system. It is known that a tyrosine residue (Tyr254) located at the C terminus of transmembrane domain (TM) V and several primarily hydrophobic amino acids present within the cytoplasmic portion of TM VI play key roles in determining the G protein coupling selectivity of the M3receptor subtype. To examine whether M3 receptor activation involves changes in the relative orientations of these functionally critical residues, pairs of cysteine residues were substituted into a modified version of the M3receptor that contained a factor Xa cleavage site within the third intracellular loop and lacked most endogenous cysteine residues. All analyzed mutant receptors contained a Y254C point mutation and a second cysteine substitution within the segment Lys484–Ser493at the intracellular end of TM VI. Following their transient expression in COS-7 cells, mutant receptors present in their native membrane environment (in situ) were subjected to mild oxidizing conditions, either in the absence or in the presence of the muscarinic agonist, carbachol. The successful formation of disulfide cross-links was monitored by studying changes in the electrophoretic mobility of oxidized, factor Xa-treated receptors on SDS gels. The observed cross-linking patterns indicated that M3receptor activation leads to structural changes that allow the cytoplasmic ends of TM V and TM VI to move closer to each other and that also appear to involve a major change in secondary structure at the cytoplasmic end of TM VI. This is the first study employing an in situdisulfide cross-linking strategy to examine agonist-dependent dynamic structural changes in a G protein-coupled receptor. |
| Databáze: |
Supplemental Index |
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