The neuronal calcium-sensor protein VILIP modulates cyclic AMP accumulation in stably transfected C6 glioma cells: amino-terminal myristoylation determines functional activity.
VILIP (visinin-like protein) is a member of the neuronal subfamily of EF-hand calcium sensor proteins. Members of this family are involved in the calcium-dependent regulation of the desensitization of signal cascades in retinal photoreceptors. To gain insight into the function of VILIP in cell signaling, we have transfected wild-type VILIP and mutant VILIP lacking the myristoylation consensus sequence into C6 glioma cells. Expression of wild-type VILIP did not significantly influence the desensitization of beta-adrenergic receptors, which are coupled to adenylyl cyclase in C6 cells. However, VILIP expression increased the beta-adrenergic receptor-stimulated cyclic AMP (cAMP) level in these cells severalfold. The stimulatory effect was also observed after direct stimulation of the adenylyl cyclase with forskolin, indicating that VILIP acts downstream of receptor and G protein in the beta-adrenergic signaling pathway in C6 cells. In contrast, the nonmyristoylated mutant of VILIP reduced cellular cAMP levels in C6 cells. Myristoylated wild-type VILIP was associated in a calcium-dependent manner with membrane fractions during subcellular fractionation, presumably owing to a calcium-myristoyl switch. In contrast, association of nonmyristoylated mutant VILIP with membranes was strongly reduced. Thus, myristoylation and most likely the calcium-dependent membrane association of VILIP are important prerequisites for the activating effect of wild-type VILIP on cAMP accumulation in C6 cells. These results suggest that VILIP acts as a calcium sensor molecule that modulates cell signaling cascades, possibly by direct or indirect regulation of adenylyl cyclase activity.
Braunewell
K H
KH
Department of Neurochemistry and Molecular Biology, Federal Institute for Neurobiology, Magdeburg, Germany.
Spilker
C
C
Behnisch
T
T
Gundelfinger
E D
ED
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000502:physiology*; D002461:Cell Line, Transformed; D000242:Cyclic AMP / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000502:physiology*; D051596:Neurocalcin; D009474:Neurons / Q000378:metabolism*; D009994:Osmolar Concentration; D051381:Rats; D044169:Receptors, Calcium-Sensing; D013329:Structure-Activity Relationship; D013347:Subcellular Fractions / Q000378:metabolism; D014018:Tissue Distribution; D014162:Transfection
D002135:Calcium-Binding Proteins; D009227:Myristic Acids; D009419:Nerve Tissue Proteins; D051596:Neurocalcin; D044169:Receptors, Calcium-Sensing; C496690:Vsnl1 protein, rat; D019814:Myristic Acid; D000242:Cyclic AMP; D002118:Calcium