Tue, Jun 28, 1994 SIGNAL TRANSDUCTION IN CLONEDnMUSCARINIC RECEPTORS Julius Axelrod National Institute of Mental Health Laboratory of Cell Biology The neurotransmitter acetylcholine can recognize two receptors. A These are nicotinic receptors that form ion channels and muscarinic receptors that are associated with $GTP(G) proteins. Since this symposium is mainly concerned with cholinergic nicotinic receptors I will confine my introductory lecture to muscarinic receptors. Two types of pharmacologic muscarinic receptors have been known for several years, Ml and M2. Ml receptors have a high affinity for perenzepine while M2 receptors have a low affinity for this drug. These pharmacologically define receptors have different biological responses and distribution. Ml receptors are localized in the brain and stimulate phosphatidyGnositoI metabolism while M2 receptors are present in the 2 Tue, Jun 28, 1994 heart and inhibit adenylate cyclase. In 1987 several groups of /t&&dgGA investigators including our laboratory genes by expression cloning A encoding five distinct muscarinic receptors. These receptors were named ml , m2, m3, m4 and m5 based on their chronological order of discovery. These receptors were found to be a member of a superfamily which span the membrane seven times and are coupled to G proteins. Using && hybridization in the rat brain, the mRNA showed heterogeneous distribution of the various muscarinic subgroups. The ml, l/&&-Q-d~b~ m3, and m4 muscarinic receptors are present in the hypocampus, striatum and dentate gyr& and are absent in the heart. The m2 receptors are present in the heart and smooth muscles but are bf low abundance in the ~4cPr'?rtd brain. The ml, and m3 receptors have been localized in the larimal, submandibular and parotid glands and the m2 and m3 receptors are present in the intestines, trachea and bladder. The localization of the m5 receptors have not been described. The ml, m2, m3, m4 and m5 cloned muscarinic receptors have been 3 Tue, Jun 28, 1994 stably transfected and expressed in a mouse A9L fibroblast cells and Chinese hamster ovary cells (CHO). Neither of these cell lines have an endogenous muscarinic receptor. These cell lines, transfected with five cloned muscarinic receptors, made it possible to examine the signal transduction pathways for each of the transfected muscarinic receptors. Carbachol,a muscarinic receptor agonist inhibited cyclic AMP in transfected ceils expressing m2 and m4, receptors. The carbachol induced inhibition was block by the muscarinic antagonist atropine. Cells e-f expressing the m3, m5 A muscarinic receptors when stimulated with carbachol generated multiple signals. These wece 1, 4, 5 inositol triphosphate (1 P3) and diacylglycerol generated from phospholipase C, arachidonic acid from phospholipase A2 and phospatidic acid from phospholipase D. These muscarinic receptors also induced cyclic AMP formation and caused an elevation of cytosolic calcium. The carbachol induced second messengers were inhibited by the atropine. Tue, Jun 28, 1994 The phospholipases activated by muscarinic receptors are differentially regulated. Phospholipase A2 and D requires calcium for activation while phospholipase C does not. Phorbol esters, compounds that activate protein kinase C, augment carbachol stimulated phospholipase A2 and arachidonic acid release. In contrast, phorbol esters pretreatment inhibitg carbachol stimulated phospholipase C, a& 6 + arbachol generates C-AMP indirectly via the activation of phospholipase &,&&&l%L c aIwnodrr/r w C. Tkn cellular calcium generated by lP3 activatesdwhich in turn activates cylic AMP. The muscarinic receptor (m3) stimulated increase in cytosolic /wGp ' calcium occurs in two phases; an initialdspike of-a&h&on followed by lower sustained increase. The initial rapid increase is caused by the release of calcium from intracellular stores by 1 P3 as a consequence of a muscarinic receptor induced stimulation of phospholipase C. This initial rise in intracellular calcium does not require the presence of extra cellular calcium. The sustained increase requires extacellular calcium and the continued presence of carbachol. In many cell types it has been Tue, Jun 28, 1994 shown that the influx of extra cellular calcium in trigger by a 1 P3 generated intr(cellular calcium. The intracellular calcium is not , - necessary for the influx of extracellular calcium &, A9L or CHO cells expressing cloned muscarinic receptors. When cells expressing the muscarinic m5 receptor were pretreated with phorbol esters a-eumpmnd * that inhibits both phospholipase C and the intracellular elevation of 1 P3 also blocked the initial rise in cytosolic calcium. However the sustained slower elevation of intracellular calcium persisted as long as carbachol was present. In the absence of extracellular calcium the carbachol stimulated influx was abolished. These findings indicated that after stimulating muscarinic receptor-j' UM+ the rapid initial rise of calcium was due to release of intracellular stores of calcium by 1 P3. The sustained elevation of calcium was the result of influx of extracellular calcium which was independent of intracellular calcium. The carbachol induced elevation of intracellular calcium was unaffected by inhibitors of voltage dependent calcium channels or high potassium depolarization. AA-- Thus it appears that receptor operated calcium channels independent of A 6 Tue, Jun 28, 1994 voltage are present in CHO and A9L fibroblasts cells. In the superfamily of seven membrane spanning receptors, the third cytosolic has been shown to represent the primary structural determinant for G-proteins. To examine the role of the third cytosolic loop in muscarinic receptors, two chimeric m2/m3 receptors were constructed in which the third cytosolic loop was exchanged between the m2 receptor and the m3 receptor in transfected A9L fibroblast cells. The wild type m2 receptor couples to inhibition of adenylate cyclase and the m3 receptors generate inositol phosphates, arachidonic acid release and cytosolic calcium elevation. Exchange of the third cytosolic loop between two receptors reverses the functional specificity of `the resultant chimeric receptors. In contrast to the wild type m2 receptor which had no effect on calcium levels, the chimeric m2 receptor having a m3 cytosolic third loop construct now cause a rapid rise in calcium levels. The chimeric m3 receptor having an m2 third loop construct failed to induce a rapid rise in calcium but stimulated the slow sustained elevation of calcium. The m3/m2 construct prevented the elevation of inositol 7 Tue, Jun 28, 1994 phosphates, arachidonic acid release and cyclic AMP generation. Thus the third cytosolic loop of the muscarinic receptors -essential for the generation of inositol phosphate, CAMP and arachidonic acid release but has no effect on the sustained calcium influx. Phospholipase C is a member of a family of several gene products that include the isozyme phospholipase Cr. Phospholipase Cy is regulated by a tyrosine kinase via a receptor operated calcium channel as shown by the following experiments. Stimulation of CHO cells expressing the m5 muscarinic receptor with carbachol resulted in an increase in tyrosine phosphorylation. The concentration dependent $imulation of tyrosine phosphorylation correlated with the concentration dependent receptor stimulation of calcium influx. Inhibitors of calcium influx blocked the muscarinic stimulation of tyrosine phoshorylation. Activation of m5 muscarinic receptorsA resulted in the tyrosine phosphorylation of phospholipase Cr. Thus the phosphorylation of Cy by muscarinic receptor activation appears to be mainly dependent on the influx of extracellular 8 calcium. Tue, Jun 28, 1994 In synaptic vesicles ATP is copackged with the neurotransmitters, acetylcholine, norepinephrine and serotonin. Upon nerve stimulation both the neurotransmitter and ATP are coreleased. ATP, in addition to its role in intermediate metabolism, can also serve as a neurotransmitter that binds to a cell surface P2 purinergic receptor. The interaction of ATP with P2 purinergic receptors stimulates phospholipase A2 to release arachidonic acid. An interaction between ATP receptors and inhibitory muscarinic receptors have been found. CHO cells have an endogenous ATP c%JJ- receptor aRel can release arachidonic acid \rrkrrn. CHO cells transfected with the inhibitory m2 or m4 r:ceptors cannot release arachidonic acid when treated with carbachol. Stimulation of CHO cells expressing the m2 or m4 receptors with carbachol together with ATP, markedly potentiated the ATP stimulated release of arachidonic acid. Treatment with atropine blocked the carbachol induced augmentation of arachidonic acid release but had no effect on ATP evoked arachidonic acid suggesting a muscarinic receptor process. Other receptors associated 9 Tue, Jun 28, 1994 t with the inhibition of adenylate cyclase including D2 dopamine, + adrenergic, and J& adenosine receptor also augmented the ATP generated arachidonic acid release. This potentiation phenomena by inhibitory receptors were blocked by pertussis toxin and inhibitor protein of kinase C suggesting the involvement of inhibitory G proteins and protein kinase C.