Muscarinic receptor-mediated calmodulin translocation in human neuroblastoma cells.
Mangels, Lori Ann
1991
Abstract
The function of the Ca$\sp{2+}$-binding protein calmodulin (CaM) is to recognize changes in intracellular Ca$\sp{2+}$ and transmit this signal inside the cell. CaM regulates enzyme systems and interacts with a host of cytoskeletal proteins in accordance with its cellular localization. This thesis examined the effects of receptor activated phosphoinositide (PPI) turnover, Ca$\sp{2+}$ fluxes and adenylyl cyclase activity on the subcellular localization of CaM and its interactions with CaM-binding proteins in SK-N-SH human neuroblastoma cells. Upon incubation of the cells with the Ca$\sp{2+}$-mobilizing agonist carbachol, a robust muscarinic receptor-specific and time-dependent translocation of CaM from membranes into the cytosol was observed. The maximal redistribution of CaM occurred at 15-30 minutes. The mechanism of the translocation involved both limbs of the PI signaling pathway since increases in intracellular Ca$\sp{2+}$ with ionomycin and protein kinase C activity with phorbol ester mimicked the effect achieved with agonist alone. Further characterization of the translocation demonstrated that CaM was released from a particulate fraction in response to carbachol and phorbol ester, and that elevated intracellular Ca$\sp{2+}$ concentrations revealed cryptic CaM in the cell cytosol. Cycloheximide had no effect on the early phase of the translocation but inhibited the elevated cytosolic CaM levels observed after 2-4 hours. Agents that increase intracellular cyclic AMP levels such as vasoactive intestinal polypeptide, dibutyryl cyclic AMP and forskolin all elicited a modest translocation of CaM from membranes into cytosol. The effects of these agents when combined with carbachol were not additive. A photoreactive CaM-diazopyruvamide derivative (CaM-DAP) was incorporated into intact cells using a scrape-loading technique to probe CaM's interactions with target proteins. Upon photochemical cross-linking, carbachol-stimulated cells displayed increased photoaffinity labeling of 70, 120 and 180 kDa adducts. The identities of target proteins were investigated using specific antisera. The SK-N-SH cells expressed a neuronotypic pattern of CaM-binding proteins. Both phosphodiesterase and calcineurin cross-linked with CaM-DAP and migrated with apparent molecular weights of 68-72 kDa. The cross-linked cytoskeletal proteins caldesmon and adducin migrated at 97 and 118 kDa, respectively. The data presented in this thesis provide evidence for receptor regulation of CaM localization. The results suggest that the translocation of CaM between cellular compartments may represent not only an amplification of the muscarinic signal, but also a means to direct the specificity of Ca$\sp{2+}$-dependent responses to agonist.Other Identifiers
(UMI)AAI9135646
Subjects
Health Sciences, Pharmacology
Types
Thesis
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