Cell Expression System in V79 Cells Transfected with Cytochrome P450 and its Performance in Metabolism Studies

The mammalian protein responsible for Ca2+ release-activated current (Icrac) may be homologous to the Drosophila protein designated trp. Thus the activity of trp, and another Drosophila protein designated trp-like or trpl, may be linked to depletion of the internal Ca2+ store via the so-called capacitative Ca2+ entry mechanism. To test this hypothesis, the effect of thapsigargin, a selective inhibitor of the endoplasmic reticulum Ca2+ pump, on trp- and trpl-induced whole cell membrane current was determined using the baculovirus Sf9 insect cell expression system. The results demonstrate that trp and trpl form Ca(2+)-permeable cation channels. The trpl encodes a nonselective cation channel that is constitutively active under basal nonstimulated conditions and is unaffected by thapsigargin, whereas trp is more selective for Ca2+ than Na+ and is activated by depletion of the internal Ca2+ store. Although evaluation of cation selectivity suggests that trp is not identical to the channel responsible for Icrac, these channels must share some structural feature(s) since both are activated by thapsigargin. A unique proline-rich region in the COOH-terminal tail of trp, which is absent in trpl, may be necessary for capacitative Ca2+ entry.

Download Full-text

[16] Functional analysis of mutant P450(C21) genes in COS cell expression system

Methods in Enzymology - Cytochrome P450 ◽

10.1016/0076-6879(91)06087-j ◽

1991 ◽

pp. 166-173 ◽

Cited By ~ 9

Author(s):

Yujiro Higashi ◽

Yoshiaki Fujii-Kuriyama

Keyword(s):

Functional Analysis ◽

Expression System ◽

Cell Expression ◽

Cell Expression System

Download Full-text

Interleukin-4 induces association of the c-fes proto-oncogene product with phosphatidylinositol-3 kinase

Blood ◽

10.1182/blood.v88.10.3910.bloodjournal88103910 ◽

1996 ◽

Vol 88 (10) ◽

pp. 3910-3918 ◽

Cited By ~ 34

Author(s):

K Izuhara ◽

RA Feldman ◽

P Greer ◽

N Harada

Keyword(s):

Expression System ◽

Sh2 Domain ◽

Pi3 Kinase ◽

Kinase Assay ◽

Interleukin 4 ◽

Related Protein ◽

Cos7 Cell ◽

Cell Expression ◽

Cell Expression System ◽

Phosphatidylinositol 3

We have previously demonstrated that interleukin-4 (IL-4) induces tyrosine phosphorylation of a protein closely related or identical to the c-fes proto-oncogene product (FES) and association of this protein with the IL-4 receptor alpha chain (IL-4R alpha). IL-4 is known to induce association of phosphatidylinositol-3 (PI3) kinase with the IL-4R alpha. Since FES contains the consensus motifs for PI3 kinase binding, we tested the possibility that FES may associate with PI3 kinase upon IL-4 stimulation. We demonstrate herein that IL-4 stimulation induced rapid association of FES or a related protein with PI3 kinase in mouse T-cell lines. We also show an association of human FES (hFES) with the src homology 2 (SH2) domain of PI3 kinase in a COS7 cell expression system. The in vitro PI3 kinase assay using COS7 cells suggested that hFES partly contributes to the association between the hIL-4R alpha and PI3 kinase. We have further identified the important region in the cytoplasmic domain of the hIL-4R alpha for association of tyrosine-phosphorylated hFES with the hIL-4R alpha and SH2 domain of PI3 kinase using a COS7 cell expression system. These results suggest that FES or a related protein/PI3 kinase pathway may play a role in the pleiotropic effects of IL-4.

Download Full-text