A heterotrimeric G protein complex couples the muscarinic m1 receptor to phospholipase C-beta (original) (raw)
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Proceedings of The National Academy of Sciences, 1990
The mechanism of phospholipase C regulation by inhibitory receptors was analyzed both in intact and in permeabilized rat thyroid cells (FRTL5). In this system, the muscarinic agonist carbachol inhibited phospholipase C, as indicated by the decrease in the basal levels of inositol 1,4,5trisphosphate as well as by the reduced adrenergic stimulation of phosphoinositol accumulation, which was paralleled by a fall in the cytosolic Ca21 levels. This inhibition involved an M2 muscarinic receptor because it was abolished by atropine but not by the Ml antagonist pirenzepine. Cells pretreated with pertussis toxin were not responsive to carbachol, indicating the involvement of a guanine nucleotide-binding protein in this inhibitory process. This possibility was further evaluated in permeabilized cells, where the carbachol inhibition was shown to be completely dependent on GTP. Known second messengers were not involved in this inhibitory process since Ca2 , cAMP, and activators of protein kinases were not able to mimic or prevent the carbachol effect either in intact or in permeabilized FRTL5 cells. In this system, the phospholipases C and A2 are coupled to two classes of muscarinic receptors that display a different sensitivity to pertussis toxin. The carbachol inhibitory effect occurred under conditions that prevented activation of phospholipase A2, excluding a role of the arachidonic acid metabolism in this process. Taken together these data provide the strongest support to date that an inhibitory guanine nucleotide-binding protein sensitive to pertussis toxin can directly mediate receptor-induced inhibition of phospholipase C.
Phosphorylation of muscarinic receptors: regulation by G proteins
Life Sciences, 1993
Effects of G proteins on the phosphorylation of muscarinic receptors (mAChRs) have been examined. Cerebral but not atrial mAChRs were phosphorylated by any one of three types of protein kinase C and 4-6 mol of phosphate were incorporated per mol of mAChR, mostly in the 12-14 kDa from the carboxyterminus. Atrial mAChRs were better substrates of cAMP-dependent protein kinase than cerebral mAChRs. Phosphorylation of mAChRs by protein kinase C or cAMP-dependent protein kinase was not dependent on the presence of agonists and G proteins except that a slight inhibition by G proteins was observed probably because G proteins were also substrates of the two kinases. Agonistdependent phosphorylation of atrial mAChRs or recombinant human mAChRs (m2 subtype) by a kinase (mAChR kinase), which is the same or very similar to 15 adrenergic receptor kinase (15ARK), was found to be regulated by the G proteins in a dual manner; stimulation by G protein ~y subunits and inhibition by G protein cc15y trimer. The inhibition by the G protein trimer is restored by addition of guanine nucleotides and is considered to be due to the formation of a ternary complex of agonist, mAChR and guanine nucleotide free G proteins. The stimulation by G protein 15Y subunits was also observed for the light-or agonist-dependent phosphorylation of rhodopsin and 15AR by the mAChR kinase but not for the lightdependent phosphorylation of rhodopsin by rhodopsin kinase. The phosphorylation by 15ARK 1 was also found to be stimulated by G protein 15v subunits. The 15v subunit is considered to interact with the extra 130 amino acid residue carboxyterminal tail of 15ARK, which does not exist in rhodopsin kinase, and the interaction results in the activation of the kinase. We may assume that the G protein coupled receptor kinase is an effector of G protein 15v subunits and that one of the functions of 15y subunits is to stimulate the phosphorylation of G protein coupled receptors thereby facilitating their desensitization.
Life Sciences, 1993
M1 muscarinic cholinergic receptors, G_ and G,~ (G ....), and phospholipase C-E1 were highl~ purifie~ fr~±~oth natural sources and cells that express the appropriate cDNA's. When the proteins were coreconstituted into phospholipid vesicles, the receptor efficiently and selectively promoted the activation of Ga/ll , leading to marked stimulation of PLC activity ~n the presence of GTPTS. No stimulation was observed in the presence of GTP, however, which led to the finding that PLC-$1 stimulates the hydrolysis of G_,ll-bOund GTP at least 50-fold. Thus, PLC-~I is a G~ase activating protein, a GAP, for its physiologic regulator G_,I-. We discuss the implications of PLC-$1's GAP ac~ivlty on the M1 muscarlnlC chollnerglc signaling pathway.
The Journal of biological chemistry, 1986
The GTP binding regulatory protein (Ni involved in adenylate cyclase inhibition was purified from rat brain and reconstituted, together with muscarinic cholinergic receptors purified from porcine brain, into phospholipid vesicles. Guanosine 5'-O-(3-[35S]thio)-triphosphate ([35S]GTP gamma S) binding and GTP hydrolyzing activities of reconstituted Ni were stimulated by the addition of a muscarinic agonist, carbachol. The effect of carbachol was to increase the Vmax values of these activities, but the Km values were also increased slightly in most cases. Carbachol bound to vesicles with the same order of magnitude of Km as that for stimulation of GTPase. The affinity of this binding was reduced by GTP gamma S, indicating that the high-affinity receptor-Ni complex was formed in a GTP-dependent manner in reconstituted vesicles. Incubation of Ni with NAD and islet-activating protein (IAP), pertussis toxin, caused ADP-ribosylation of the alpha-subunit of Ni. The criteria for the recept...
Differential agonist-induced regulation of human M2 and M3 muscarinic receptors
Biochemical Pharmacology, 2003
We have compared the regulation of M 2 and M 3 muscarinic receptors heterologously expressed in HEK-293 cells upon long-term exposure towards the agonist carbachol. Carbachol time-and concentration-dependently reduced M 2 receptor density with a maximum reduction of about 60%. Treatment with 1 mM carbachol for 24 hr was accompanied by desensitisation of carbachol-induced Ca 2þ elevations (maximum response reduced by 70%) but not by alterations in the expression of various G-protein a-subunits. Consistently, heterologous desensitisation of Ca 2þ elevations by the purinergic receptor agonist ATP or by sphingosine-1-phosphate was not detected. In contrast, carbachol time-and concentration-dependently up-regulated M 3 receptors with maximum increases to about 350% of control values. The up-regulation was fully blocked by cycloheximide indicating that it was dependent on protein synthesis. Concomitant with the up-regulation of the M 3 receptor was a reduction in the expression of the a-subunit of G q/11. The net effect of these two opposite regulatory mechanisms was a lack of alteration of carbachol-stimulated Ca 2þ elevation. However, the reduction of G q/11 was accompanied by a heterologous desensitisation of Ca 2þ elevations by ATP and sphingosine-1-phosphate. Levels of M 2 and M 3 receptor mRNA as assessed by real-time PCR were not significantly altered by carbachol exposure for either receptor, suggesting that alterations of mRNA stability did not contribute to the observed changes in receptor number. We conclude that M 2 and M 3 receptor expression within the same cell undergoes differential agonist-induced regulation being accompanied by distinct regulation of G-protein expression leading to differential effects on signal transduction by other receptor systems.
Current Biology, 1995
Background: One of the principal mechanisms by which G-protein-coupled receptors evoke cellular responses is through the activation of phospholipase C (PLC) and the subsequent release of Ca 2+ from intracellular stores. Receptors that couple to pertussis toxin (PTX)-insensitive G proteins typically evoke large increases in PLC activity and intracellular Ca 2 + release. In contrast, receptors that use only PTX-sensitive G proteins usually generate weak PLC-dependent responses, but efficiently regulate a second effector enzyme, adenylyl cyclase. For example, in many cell types, agonist binding by the m4 muscarinic acetylcholine receptor (m4 receptor) results in a strong inhibition of adenylyl cyclase and very little stimulation of PLC activity or release of intracellular Ca 2+. We have investigated whether the weak, PTX-sensitive stimulation of PLC activity by the m4 receptor can play a significant role in the generation of cellular responses. Results: We report here that PTX-sensitive Ca 2+ release mediated by the m4 receptor in transfected Chinese hamster ovary cells is greatly enhanced when endogenous purinergic receptors simultaneously activate a PTX-insensitive signaling pathway. Furthermore, m4-receptor-induced transcription of the c-fos gene (a Ca 2 +-sensitive response) is similarly potentiated when purinergic receptors are coactivated. These enhanced m4-receptor-dependent Ca 2 + responses do not require an influx of external Ca 2+ , and occur in the absence of detectable purinergic-receptorstimulated Ca 2+ release; they apparently require the activation of both PTX-sensitive and PTX-insensitive G-protein pathways. Measurements of phosphoinositide hydrolysis indicate that the enhancement of m4-receptormediated Ca 2 + signaling by purinergic receptors is due to a synergistic increase in agonist-stimulated PLC activity. Conclusions: These studies demonstrate that the potency of m4-receptor-mediated PLC signaling is highly dependent upon the presence or absence of other PLCactivating agonists. The ability of the m4 receptor to evoke a strong, but conditional, activation of PLC may allow this type of receptor to participate in a coincidence-detection system that amplifies simultaneous PLC-activating signals through a mechanism involving crosstalk between PTXsensitive and PTX-insensitive G-protein pathways.
FEBS Letters, 1996
nic m3 receptor plays a dominant role in the regulation of Abstract The m2 and m3 muscarinic acetylcholine receptors Ca 2+ influx in CHO cells. were expressed in CHO cells and were shown to couple to the release of calcium from intracellular stores. The m3 receptor, but not the m2 receptor, also coupled to calcium influx. Chimeric m21 2. Materials and methods m3 receptors were used to determine the structural domain of the m3 receptor linked to the regulation of calcium influx. It was 2.1. Chimeric eDNA construct preparation and cell culture found that the third intracellular loop of m3 receptor plays a