title: The C-terminal tail of the M3-muscarinic receptor possesses anti-apoptotic properties.
abstract: This study investigates the mechanisms by which the muscarinic receptor gene family can protect against apoptosis. Chinese hamster ovary cells transfected with human muscarinic receptor subtypes underwent apoptotic cell death following treatment with the DNA-damaging agent etoposide. Apoptosis was significantly reduced following muscarinic receptor stimulation of cells that were transfected with receptor subtypes that couple to the Gq/11/phospholipase C pathway, namely M1, M3, and M5. No protection was detected in cells transfected with the Gi-coupled M2 and M4 receptors. Further analysis of the Gq/11-coupled M3 receptor revealed that truncation of the carboxyl-tail (Delta 565-M3 mutant) removed the ability of the receptor to protect against etoposide-induced cell death. This mutation did not affect the ability of the receptor to signal through the phospholipase C pathway. Furthermore, activation of the Delta 565-M3 receptor resulted in robust activation of the extracellular-regulated kinase (ERK) and c-Jun kinase (JNK). The Delta 565-M3 receptor mutant also underwent agonist-driven phosphorylation in a similar manner to the wild-type receptor indicating that the anti-apoptotic effect of the M3 receptor is independent of receptor phosphorylation. Consistent with this was the fact that two M3-muscarinic receptor mutants deficient in agonist-induced receptor phosphorylation were capable of producing a full anti-apoptotic response. We conclude that the anti-apoptotic response of the muscarinic receptor family was confined to the Gq/11-coupled members of this family. The direct involvement of Gq/11/phospholipase C signaling and the ERK-1/2 and JNK pathways together with receptor phosphorylation in the anti-apoptotic response were eliminated. Mutation of a poly-basic region within the short C-terminal tail of the M3-muscarinic receptor inhibited the ability of the receptor to induce an anti-apoptotic response. We conclude that the conserved poly-basic region in the C-terminal tail of the M1, M3, and M5 receptors contributes to the ability of these receptors to mediate protection against apoptotic cell death.
authors.affiliation: Department of Cell Physiology & Pharmacology, Medical Sciences Building, University of Leicester, University Road, Leicester LE1 9HN, United Kingdom. dcb8@le.ac.uk
authors.forename: David C
authors.initials: DC
authors.lastname: Budd
authors.forename: John
authors.initials: J
authors.lastname: McDonald
authors.forename: Nita
authors.initials: N
authors.lastname: Emsley
authors.forename: Kelvin
authors.initials: K
authors.lastname: Cain
authors.forename: Andrew B
authors.initials: AB
authors.lastname: Tobin
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects; D016466:CHO Cells; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D006224:Cricetinae; D004249:DNA Damage; D004789:Enzyme Activation / Q000187:drug effects; D005047:Etoposide / Q000494:pharmacology; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins / Q000502:physiology; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D008856:Microscopy, Fluorescence; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D010766:Phosphorylation; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D016415:Sequence Alignment; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism
chemical_list: D017304:Annexin A5; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic; D005047:Etoposide; D048031:JNK Mitogen-Activated Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D020929:Mitogen-Activated Protein Kinase Kinases; D010738:Type C Phospholipases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins; D016650:Fluorescein-5-isothiocyanate