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Structure/Function Relationships of a G-Protein Coupling Pocket Formed by the Third Intracellular Loop of the m5 Muscarinic Receptor
Using random saturation mutagenesis, we have previously identified the amino acids K439, A440, and A441 in the C-terminus of the third intracellular loop (Ci3) of the m5 muscarinic receptor as being critical for G-protein coupling [Burstein, E. S., Spalding, T. A., Hill-Eubanks, D., and Brann, M. R....
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Published in: | Biochemistry (Easton) 1998-03, Vol.37 (12), p.4052-4058 |
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Main Authors: | , , |
Format: | Article |
Language: | English |
Subjects: | |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
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Summary: | Using random saturation mutagenesis, we have previously identified the amino acids K439, A440, and A441 in the C-terminus of the third intracellular loop (Ci3) of the m5 muscarinic receptor as being critical for G-protein coupling [Burstein, E. S., Spalding, T. A., Hill-Eubanks, D., and Brann, M. R. (1995) J. Biol. Chem. 270, 3141−3146]. In the present study, we have constructed a series of point mutants at each of these residues and characterized their functional phenotypes in order to define the structure/function relationships of each of these residues for G-protein coupling. Although a wide variety of substitutions were tolerated at K439, most caused significant increases in the EC50 of carbachol and decreases in the maximum response (R max). Only other basic residues were well tolerated (70% of wild type). Acidic substitutions had the largest effects, reducing R max to under 20% of wild type. At A440, only the conservative substitution threonine was well tolerated. Substitutions by hydrophobic, polar, and basic residues caused 10−80-fold increases in EC50 values and in many cases also significantly reduced R max ( |
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ISSN: | 0006-2960 1520-4995 |
DOI: | 10.1021/bi972132j |