The Neuronal Calcium‐Sensor Protein VILIP Modulates Cyclic AMP Accumulation in Stably Transfected C6 Glioma Cells: Amino‐Terminal Myristoylation Determines Functional Activity

Abstract

VILIP (Mjsinin-like protein) is a member of the neuronal subfamily of EF-hand calcium sensor proteins. Members of thisfamily 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 VI LIP lacking the myristoylation consensus sequence into C6 glioma cells. Expression of wildtype VILIP did not significantly influence the desensitization of /3-adrenergic receptors, which are coupled to adenylyl cyclase in C6 cells. However, VILIP expression increased the~-adrenergicreceptor-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 /3-adrenergic signaling pathway in C6 cells. In contrast, the nonmyristoylated mutant of VILIP reduced cellular cAMP levels in 06 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 06 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.

Neuronal Ca2+ sensor protein VILIP-1 affects cGMP signalling of guanylyl cyclase B by regulating clathrin-dependent receptor recycling in hippocampal neurons

“…Previous results suggested the NCS protein VILIP-1 as a modulator of several neuronal signalling cascades: it has been implicated in modulation of adenylyl cyclase activity (Braunewell et al, 1997;Lin et al, 2002a;Mahloogi et al, 2003) and related cyclase enzymes, including the soluble and the membrane-localized GCs Spilker et al, 2002b). Interestingly, these data were recently supported by findings that the related protein neurocalcin δ was found to interact with and directly influence related guanylyl cyclases, namely the retinal and the olfactory GCs Duda et al, 2004).…”

“…Hippocampal neurons were transfected with cDNA coding for GFP, VILIP-1-or VILIP-3-GFP after 6 days in culture essentially as described (Spilker et al, 2002a;Spilker et al, 2002b;Spilker and Braunewell, 2003) using the lipofectamin transfection method (Invitrogen). ) in a humidified 95% air, 5% CO 2 incubator essentially as described previously (Braunewell et al, 1997). In C6-V1 and C6-V1M cells, VILIP-1-expression was induced overnight with 5 mM IPTG and, by virtue of leaky expression of VILIP-1 in C6-V1 and C6-V1M cells, these cells were compared with parental C6 cells.…”

“…Nuclei and debris were removed by centrifugation at 1000 g for 5 minutes and equal protein concentrations (in µg protein ml -1 buffer) of the resulting cell homogenates were applied to 5-20% gradient SDS-PAGE. To investigate the expression levels of VILIP-1 and GC-B in the cells the previously described antibodies were used for western blot analysis (Braunewell et al, 1997). Protein concentrations were measured by the BCA assay (Pierce, Rockford, IL).…”

The GDNF Target Vsnl1 Marks the Ureteric Tip

“…Vsnl1 belongs to the neuronal calcium sensor protein family that modulates calcium-dependent cell signaling events. 46 Activation of ERK-MAPK pathway by GDNF/Ret is a calcium-dependent process essential to cell proliferation. 47,48 The dp-ERK is an established reporter of Ret signaling, 40,41 with an uneven expression in the UB.…”

N-terminal Myristoylation Regulates Calcium-induced Conformational Changes in Neuronal Calcium Sensor-1

“…For example, the association of p22 with microtubules is dependent both on Ca 2ϩ and myristoylation (15), whereas in GCAP1, GCAP2, and VILIP, it is just the Ca 2ϩ binding that regulates their function (16,17). Hippocalcin, neurocalcin, and NCS-1, all of Ca 2ϩ sensor family, use their myristoyl group differently.…”