P. Hogger et al., ACTIVATING AND INACTIVATING MUTATIONS IN N-TERMINAL AND C-TERMINAL I3LOOP JUNCTIONS OF MUSCARINIC ACETYLCHOLINE HM1 RECEPTORS, The Journal of biological chemistry, 270(13), 1995, pp. 7405-7410
The N- and C-terminal junctions of the third intracellular loop (i3) o
f G protein coupled receptors play a role in the coupling process, We
had previously constructed two triple point alanine mutants of the i3
junction of the muscarinic Hm1 receptor, W209A/I211A/Y212A and E360A/K
362A/T366A, which are defective in mediating carbachol stimulation of
phosphatidylinositol (PI) turnover (Moro, O., Lameh, J., Hogger, P., a
nd Sadee, W. (1993) J. Biol. Chem. 268, 22273-22276). Each of the corr
esponding six single point mutations were constructed to determine res
idues crucial to receptor coupling. Mutants W209A and T366A were simil
ar to or only slightly less effective than wild type Hm1 in stimulatin
g PI turnover. In the N-terminal junction, I211A and Y212A were defect
ive in coupling, and I211A was even more defective than the correspond
ing triple mutant, Therefore, the triple mutation compensated at least
partially for the effect of these two single point mutations. In the
C-terminal i3 loop junction, mutant K362A was again more strongly defe
ctive than the corresponding triple mutant, In contrast, mutation E360
A was found to be activating, leading to elevated PI turnover in the a
bsence of agonist and sensitization toward carbachol activation. Activ
ating mutations in the C terminal i3 loop junction have been reported
previously for the adrenergic receptors, but E360A represents the firs
t muscarinic receptor with substantial basal activity, The effects of
the single point mutations observed in this study were not readily pre
dictable from similar mutations from closely related G protein-coupled
receptors despite sequence conservation in the is loop junctions. Our
results caution against defining precise coupling domains in these re
gions by mutagenesis results.