G protein coupling to M1 and M3 muscarinic receptors in sublingual glands

Abstract

Rat sublingual gland M1 and M3 muscarinic receptors each directly activate exocrine secretion. To investigate the functional role of coreceptor expression, we determined receptor-G protein coupling. Although membrane proteins of 40 and 41 kDa are ADP-ribosylated by pertussis toxin (PTX), and 44 kDa proteins by cholera toxin (CTX), both carbachol-stimulated high-affinity GTPase activity and the GTP-induced shift in agonist binding are insensitive to CTX or PTX. Carbachol enhances photoaffinity labeling ([α-32P] GTP-azidoaniline) of only 42-kDa proteins that are subsequently tractable to immunoprecipitation by antibodies specific for Gαq or Gα11 but not Gα12 or Gα13. Carbachol-stimulated photoaffinity labeling as well as phosphatidyhnositol 4,5-bisphosphate (PIP2) hydrolysis is reduced 55% and 60%, respectively, by M1 receptor blockade with ml-toxin. Gαq/11-specific antibody blocks carbachol-stimulated PIP2 hydrolysis. We also provide estimates of the molar ratios of receptors to Gαq and Gα11. Although simultaneous activation of M1 and M3 receptors is required for a maximal response, both receptor subtypes are coupled to Gαq and Gα11 to stimulate exocrine secretion via redundant mechanisms.