Futter, Marie; (2000) Muscarinic receptor activation of the MAP kinase signalling pathway and its physiological consequences. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

A functional cholinergic system and MAPK are found to be necessary for learning and memory processes, however the molecular basis for this is poorly understood. Therefore, mAChR activation of ERK1 and ERK2 has been studied in primary cortical neurones and in a model system, a COS-7 cell line overexpressing the different mAChR subtypes (M1-M4). In primary cortical neurones ERK1/2 are activated by the muscarinic agonist carbachol in a dose- and time- dependent manner, peaking at 30 minutes and lasting at least 2 hours. In COS-7 cells expressing the different mAChR subtypes ERK1/2 activation was induced more rapidly, peaking within 5 minutes and lasting at least 2 hours. ERK1/2 activation in COS-7 cells was dose-dependent. The four mAChR subtypes had different efficacies for the activation of ERK1/2 in the order, M1>M3>M2>M4. Inhibitors of key mediators in the MAPK signalling pathway have been studied. In COS-7 cells, pertussis toxin, an inhibitor of receptor stimulated G-protein activation which selectively inhibits Gi/o but not Gq/11, attenuates ERK activation by M1, M2 and M4 but not, apparently, M3. ERK1/2 activation by each of the four different mAChR subtypes studied, was Src- and EGFR- dependent and partially PI3K- and Ca2+-dependent. With the exception of M4, mAChR stimulated ERK1/2 activation was independent of PKC. In primary neurones ERK1/2 activation was Src-dependent, partially EGFR- and PI3K-dependent and Ca2+- and PKC-independent. Knock out mice for Fyn, a member of the Src family of tyrosine kinases and ErbB4, a member of the EGFR family of receptor tyrosine kinases, had a fully functional MAPK response. Alanine mutants of transmembrane spanning helix seven (TM7) of the M1 mAChR have been studied. Previous data from this laboratory demonstrate that the residues N414, P415 and Y418 from the highly conserved signature sequence, NPXXY, are important for a functional phosphoinositide response. Preliminary results suggest that these residues are also important for the activation of the MAPK response. In addition three other residues, T412, V413 and L420 may also be involved in the activation of ERK1/2.

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