Coupling of M₃ Acetylcholine Receptor to Gq₁₆ Activates a Natriuretic Peptide Receptor Guanylyl Cyclase

Abstract

Muscarinic activation of tracheal smooth muscle (TSM) involves a M₃AChR/heterotrimeric-G protein/NPR-GC coupling mechanism. G protein activators Mastoparan (MAS) and Mastoparan-7 stimulated 4- and 10-fold the NPR-GC respectively, being insensitive to PTX and antibodies against Gα_i/o subfamily. Muscarinic and MAS stimulation of NPR-GC was blocked by antibodies against C-terminal of Gα_q16, whose expression was confirmed by RT-PCR. However, synthetic peptides from C-terminal of Gα_q15/16 stimulated the NPR-GC. Coupling of α_q16 to M₃AChR is supported by MAS decreased [³H]QNB binding, being abolished after M₃AChR-4-DAMP-alkylation. Anti-i₃M₃AChR antibodies blocked the muscarinic activation of NPR-GC, and synthetic peptide from i₃M₃AChR (M₃P) was more potent than MAS increasing GTPγ [³⁵S] and decreasing the [³H]QNB activities. Coupling between NPR-GC and Gα_q16 was evaluated by using trypsin-solubilized-fraction from TSM membranes, which displayed a MAS-sensitive-NPR-GC activity, being immunoprecipitated with anti-Gα_q16, also showing an immunoreactive heterotrimeric-G-β -subunit. These data support the existence of a novel transducing cascade, involving Gα_q16β γ coupling M₃AChR to NPR-GC.

Key Words: :

• NPR-B
• G proteins
• Muscarinic receptors
• Guanylyl cyclases
• Tracheal smooth muscle

Abbreviations
ANP,	=	atrial NP 1-28;
BTSM,	=	bovine tracheal smooth muscle;
CC, Carbamylcholine, CNP,	=	C-type NP 1-22;
GC,	=	guanylyl cyclase;
GC-A,	=	ANP-sensitive guanylyl cyclase;
GC-B,	=	CNP-sensitive guanylyl cyclase;
GC-C,	=	Escherichia coli enterotoxin/guanylin-sensitive guanylyl cyclase;
IBMX,	=	Isobutyl-methyl-xantine;
i2M3AChR,	=	second intracellular loop of M3AChR;
i3M3AChR,	=	third intracellular loop M3AChR;
i3 M2AChR,	=	third intracellular loop M2AChR;
MAS, Mastoparan, MAS-7 and MAS-17,	=	7-mer and 17-mer analogs of Mastoparan;
M3P and M2P,	=	peptide fragments of i3 loop of M3 and M2AChR respectively;
mGC,	=	membrane-bound Guanylyl Cyclase;
NP,	=	natriuretic peptide;
NPR-GC, natriuretic peptide-sensitive Receptor Guanylyl Cyclase, ODQ,	=	1H-[1,2,4] oxodiazolo[4,3-] quinoxalin-1-one;
PTX,	=	Pertussis toxin.

Abbreviations
ANP,	=	atrial NP 1-28;
BTSM,	=	bovine tracheal smooth muscle;
CC, Carbamylcholine, CNP,	=	C-type NP 1-22;
GC,	=	guanylyl cyclase;
GC-A,	=	ANP-sensitive guanylyl cyclase;
GC-B,	=	CNP-sensitive guanylyl cyclase;
GC-C,	=	Escherichia coli enterotoxin/guanylin-sensitive guanylyl cyclase;
IBMX,	=	Isobutyl-methyl-xantine;
i2M3AChR,	=	second intracellular loop of M3AChR;
i3M3AChR,	=	third intracellular loop M3AChR;
i3 M2AChR,	=	third intracellular loop M2AChR;
MAS, Mastoparan, MAS-7 and MAS-17,	=	7-mer and 17-mer analogs of Mastoparan;
M3P and M2P,	=	peptide fragments of i3 loop of M3 and M2AChR respectively;
mGC,	=	membrane-bound Guanylyl Cyclase;
NP,	=	natriuretic peptide;
NPR-GC, natriuretic peptide-sensitive Receptor Guanylyl Cyclase, ODQ,	=	1H-[1,2,4] oxodiazolo[4,3-] quinoxalin-1-one;
PTX,	=	Pertussis toxin.