Analysis of the interacting partners of the neuronal calcium‐binding proteins L‐CaBP1, hippocalcin, NCS‐1 and neurocalcin δ

Abstract

Intracellular Ca2+ signals are transduced by the binding of Ca2+ to sensor proteins, which subsequently modify the activity of their target proteins. Identification of these target proteins is, therefore, important for an understanding of cellular signalling processes. We have investigated the binding partners of four EF-hand Ca2+-binding proteins. Three proteins of the neuronal calcium sensor (NCS) family, hippocalcin, NCS-1 and neurocalcin delta were prepared as N-terminally tagged GST fusion proteins, and the less closely related protein L-CaBP1 was prepared in both N- and C-terminally tagged forms, the latter requiring generation of a new vector. Immobilised fusion proteins were used to purify binding partners from bovine brain cytosol and membrane extracts in the presence of 1 microM free Ca2+. Bound proteins were eluted with Ca2+-free and high-salt buffers and eluted proteins were identified by MALDI-MS and Western blotting. New protein targets detected included ARF1, Ca2+-dependent activator protein for secretion 1, cyclic nucleotide 3',5'-phosphodiesterase, the vacuolar ATPase, AP1 and AP2 complexes and the type I TGF-beta receptor. While certain of these interactions occurred with more than one of the Ca2+-binding proteins, others were found to be specific targets for particular Ca2+ sensors, and many of these did not overlap with known calmodulin-binding proteins. These findings provide new clues to the functional roles of the neuronal calcium sensor proteins.

Whole genome expression profiling of blood cells in ovarian cancer patients -Prognostic impact of theCYP1B1,MTSS1,NCALD, andNOP14genes

“…A study by Couvelard et al found NCALD gene expression to be one of many genes that can distinguish between metastatic and non-metastatic pancreatic endocrine tumor tissue [23]. However, another gene belonging to the same gene family, the neuronal Ca 2+ sensor protein family (NCS), termed VILIP1 [24], has been more extensively studied in cancer, and shown to act as a tumor suppressor gene by inhibiting cell proliferation, adhesion, and invasiveness [25,26]. The VILIP-1 protein and mRNA was down-regulated in a study on non-small cell lung carcinoma [25], and high gene expression was reported to be associated with a high rate of lymph node metastasis and poor prognosis in colorectal cancer patients [27].…”

The Diversity of Calcium Sensor Proteins in the Regulation of Neuronal Function

“…Other NCS-1 target proteins (Fig. 3) appear to be specific for NCS-1 (Haynes et al 2006). Various studies have implicated NCS-1 in the regulation of VGCCs (Weiss et al 2000;Tsujimoto et al 2002;Dason et al 2009) but there is as yet no evidence for a direct interaction.…”

“…Many different binding partners have been identified for NCS-1 (Haynes et al 2006;Haynes et al 2007) (Fig. 3) and in some cases these interactions overlap with those of calmodulin (Schaad et al 1996).…”

Demonstration of Binding of Neuronal Calcium Sensor-1 to the Cav2.1 P/Q-Type Calcium Channel

“…43,44 NCS-1 exhibits enriched expression in mammalian brain tissue, 45 and many of the aforementioned functions are related to neuronal activity. NCS-1 interacts with a number of targets in common with CaM, 24 and a previous functional study identified links between NCS-1 and CDF of P/Q-type channel activity in rat primary neurons. 20 Other studies have additionally indicated a cellular connection between NCS-1 and P/Q-type channel activity in bovine adrenal chromaffin cells 21,22 and Drosophila .…”