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Title: The activation of guanine nucleotide binding proteins by muscarinic acetylcholine receptor subtypes
Author: Akam, Elizabeth Claire
Awarding Body: University of Leicester
Current Institution: University of Leicester
Date of Award: 1999
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Agonist-stimulation of human recombinant M1, M2, M3 and M4 receptors, expressed in Chinese hamster ovary cells, was investigated at the level of G protein activation. Functional responses were determined by a number of methods including [35S]-GTPS binding in membranes using both filtration-based and immunoprecipitation-based procedures: Ins(1,4,5)P3 accumulation and 45Ca2+ release from permeabilised cell suspensions; and cAMP accumulation in cell suspensions. M2 and M4 receptors, with equivalent expression levels in this recombinant system, were found only to couple to pertussis toxin-sensitive G proteins with near equal kinetics. Methacholine appeared equipotent when activating the total G protein complement through the M2 and M4 receptors, however, it appeared more potent when activating Gi3/o through the M2 compared to the M4 muscarinic receptor. Using equivalent expression levels of M1 and M3 receptors both the subtypes were found to couple to both pertussis toxin-sensitive and -insensitive G proteins. CHO-M1 and -M3 mediated Ins(1,4,5)P3 generation after pertussis toxin pre-treatment suggested the functional significance of coupling to multiple G protein classes may be in the stimulation of PLC by -subunits derived from Gi-like G proteins. The activation of Gq/11 through the M1 receptor subtype, after methacholine-stimulation, is faster, greater and more potent than that mediated by the M3 receptor subtype, suggesting that the intrinsic activity of the M1 subtype is greater than that of the M3 subtype. The 'partial' agonist pilocarpine also displayed very different G protein activation profiles after stimulation of M1, M2, M3 and M4 receptor subtypes, suggesting that agonists acting at different receptor subtypes may be capable of inducing relatively selective coupling of the occupied receptor to available G proteins. This study therefore concludes that muscarinic receptor subtypes display divergent G protein activation profiles after either 'full' or 'partial' agonist-stimulation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available