Cell signalling in Chinese hamster ovary cells expressing recombinant muscarinic receptors.
thesisposted on 19.11.2015, 08:43 by Neil Thornton. Burford
Agonist stimulation of recombinant m1, m2, and m3 muscarinic receptors, expressed in Chinese hamster ovary (CHO) cells, was compared. Carbachol binding affinity, and its modification by cations, guanine nucleotides and PTX pretreatment, was compared in washed membrane preparations of each of the CHO cell clones. Functional responses, determined by carbachol stimulation, were: [35S]GTPS binding in membranes; Ins(l,4,5)P3 accumulation in intact cells; 45ca2+ release from permeabilized cells; and cAMP accumulation in intact cells. m2-Transfected CHO cells were found to couple to AC, mediating inhibition of forskolin-stimulated cAMP accumulation, via PTX-sensitive G proteins. After PTX pretreatment of these cells, carbachol mediated a small potentiation of forskolin-stimulated cAMP accumulation, though with a much lower carbachol potency compared with the inhibitory response. m1 and m3-transfected CHO cell clones were found to couple with both PTX-sensitive and PTX-insensitive G proteins, at relatively high levels of receptor expression. The PTX- insensitive G proteins mediated agonist-stimulated PLC activation and were involved in the activation of AC (though at a much lower potency). The mechanism, by which m1, m2 and m3 muscarinic receptors stimulated AC activity was not thought to be due to crosstalk via PLC activity. The level of m3 muscarinic receptor expression, in CHO cells, was found to markedly affect both the potency, and the maximal responsiveness, with which carbachol mediated PLC activation and AC activation. Furthermore, at lower levels of receptor expression, m3 muscarinic receptors appeared to couple to a lesser extent with PTX-sensitive G proteins. The study, therefore, concluded that comparisons of agonist-mediated responses between muscarinic receptor subtypes, expressed in CHO cells, must be performed at similar levels of receptor expression. At similar receptor densities, m1 and m3 muscarinic receptors, in CHO cells, produced very similar responses to carbachol.