Cleavage-resistant fusion proteins of the M(2) muscarinic receptor and Gα(i1). Homotropic and heterotropic effects in the binding of ligands
| Autor: | James W. Wells, Amy W.-S. Ma, Dengbo Ma, John Y. Dong |
|---|---|
| Rok vydání: | 2010 |
| Předmět: |
G protein
Recombinant Fusion Proteins Molecular Sequence Data Biophysics Cooperativity Plasma protein binding CHO Cells GTP-Binding Protein alpha Subunits Gi-Go Ligands Biochemistry Cell Line 03 medical and health sciences Cricetulus Cricetinae Animals Humans Amino Acid Sequence Receptor Molecular Biology Cells Cultured 030304 developmental biology G protein-coupled receptor 0303 health sciences Receptor Muscarinic M2 Chemistry Chinese hamster ovary cell 030302 biochemistry & molecular biology Muscarinic acetylcholine receptor M2 Fusion protein Sequence Alignment Protein Binding |
| Zdroj: | Biochimica et biophysica acta. 1810(6) |
| ISSN: | 0006-3002 |
| Popis: | Background G protein-coupled receptors fused to a Gα-subunit are functionally similar to their unfused counterparts. They offer an intriguing view into the nature of the receptor–G protein complex, but their usefulness depends upon the stability of the fusion. Methods Fusion proteins of the M2 muscarinic receptor and the α-subunit of Gi1 were expressed in CHO and Sf9 cells, extracted in digitonin–cholate, and examined for their binding properties and their electrophoretic mobility on western blots. Results Receptor fused to native αi1 underwent proteolysis near the point of fusion to release a fragment with the mobility of αi1. The cleavage was prevented by truncation of the α-subunit at position 18. Binding of the agonist oxotremorine-M to the stable fusion protein from Sf9 cells was biphasic, and guanylylimidodiphosphate promoted an apparent interconversion of sites from higher to lower affinity. With receptor from CHO cells, the apparent capacity for N-[3H]methylscopolamine was 60% of that for [3H]quinuclidinylbenzilate; binding at saturating concentrations of the latter was inhibited in a noncompetitive manner at low concentrations of unlabeled N-methylscopolamine. Conclusions A stable fusion protein of the M2 receptor and truncated αi1 resembles the native receptor–G protein complex with respect to the guanyl nucleotide-sensitive binding of agonists and the noncompetitive binding of antagonists. General significance Release of the α-subunit is likely to occur with other such fusion proteins, rendering the data ambiguous or misleading. The properties of a chemically stable fusion protein support the notion that signaling proceeds via a stable multimeric complex of receptor and G protein. |
| Databáze: | OpenAIRE |
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