Inositol 5'-phosphatase, SHIP1 interacts with phospholipase C-gamma1 and modulates EGF-induced PLC activity

Phospholipase C-gamma1, containing two SH2 and one SH3 domains which participate in the interaction between signaling molecules, plays a significant role in the growth factor-induced signal transduction. However, the role of the SH domains in the growth factor-induced PLC-gamma1 regulation is unclear. By peptide-mass fingerprinting analysis, we have identified SHIP1 as the binding protein for the SH3 domain of PLC-gamma1. SHIP1 was co-immunoprecipitated with PLC-gamma1 and potentiated EGF-induced PLC-gamma1 activation. However, inositol 5'-phosphatase activity of SHIP1 was not required for the potentiation of EGF-induced PLC-gamma1 activation. Taken together, these results suggest that SHIP1 may function as an adaptor protein which can potentiate EGF-induced PLC-gamma1 activation without regards to its inositol 5'-phosphatase activity.

The extracellular calcium sensing receptor is expressed in mouse mesangial cells and modulates cell proliferation

The extracellular calcium sensing receptor (CaSR) belongs to the type III family of G-protein-coupled receptors, a family that comprises the metabotropic glutamate receptor and the putative vomeronasal organ receptors. The CaSR plays an important role for calcium homeostasis in parathyroid cells, kidney cells and other cells to directly 'sense' changes in the extracellular calcium ion concentration ((Ca2+)o). The mesangial cells are known to be involved in many pathologic sequences through the mediation of altered glomerular hemodynamics, cell proliferation, and matrix production. In this study, we examined the expression of the CaSR in the mouse mesangial cell lines (MMC, ATCC number CRL-1927). Reverse transcription- polymerase chain reaction (RT-PCR) was perform with CaSR-specific primers, and this was followed by nucleotide sequencing of the amplified product; this process identified the CaSR transcript in the MMCs. Moreover, CaSR protein was present in the MMCs as assessed by Western blot and immunocytochemical analysis using a polyclonal antibody specific for the CaSR. Functionally, (Ca2+)o induced the increment of the intracellular calcium concentration ((Ca2+)i) in a dose-dependent manner. This (Ca2+)i increment by (Ca2+)o was attenuated by the pretreatment with a phospholipase C inhibitor (U73122) and also by a pretreatment with a CaSR antagonist (NPS 2390). The similar results were also obtained in IP3 accumulation by (Ca2+)o. To investigate the physiological effect of the CaSR, the effect of the (Ca2+)o on cell proliferation was studied. The increased (Ca2+)o (up to 10 mM) produced a significant increase in the cell numbers. This mitogenic effect of (Ca2+)o was inhibited by the co-treatment with a CaSR antagonist. From these results, the (Ca2+)o-induced (Ca2+)i elevation in the MMC is coupled with the extracellular calcium sensing receptor. Furthermore, (Ca2+)o produces a mitogenic effect in MMCs.

Purification and characterization of receptors for myoinositol trisphosphate and myoinositol tetrakis phosphate from Entamoeba histolytica.

The microsomal fraction from the log phase of Entamoeba histolytica cells contains Ins(1,4,5)P3 and Ins(1,3,4,5)P4 binding activity. The binding proteins/receptors for both Ins(1,4,5)P3 and Ins(1,3,4,5)P4 were purified and found to be specific for each ligand. The molecular masses for native proteins for InsP3 and InsP4 are 138 kDa and 130 kDa respectively having subunits of 69 kDa and 64 kDa respectively. That these proteins are associated with Ca2+ release was confirmed by including these proteins separately in proteoliposomes and adding InsP3 and InsP4 in both the cases.

Efecto del Inositol 1, 4, 5 Trifosfato sobre [Ca2+] en músculos esqueléticos de sujectos susceptibles al síndrome de hipertemia maligna / Effects of Inositol 1, 4, 5 Triphosphate on free myoplasmic calcium concentration in malignante hyperthemia suceptible patients

Se estudió el efecto del inositol 1, 4, 5 trifosfato (InsP3) sobre la concentración de Ca++ libre de mioplásmico ([Ca++]) en biopsias musculares de sujetos sanos y susceptibles a hipertermia maligna (HM) con o sin encubación previa en dantrolene. El material utilizado incluyó microeléctrodos específicos para Ca++, y la microinyección de InsP3 por separado. En fibras musculares no susceptibles a HM, la inyección de 0,5 µM de InsP3 indujo un aumento de la [Ca++], de 0,12 ñ 0.01 µM a 0,37 ñ 0,03 µM que lle gó a 0,81 ñ 0,04 µM al doblar la dosis, con oscilación sarcométrica en casi la mitad de las preparaciones con esta dossis. El mismo procedimiento en miofibrillas susceptibles a HM produjo efectos mayores y más sostenidos; con 0,5 µM de InsP3 [Ca++], basal de 0,32 ñ 0,02 µM se elevó a 1,22 ñ 0,08 µM, y duplicando las dosis sólo se pudo medir algunas células que no sufrieron contracción local, que de 0,36 ñ 0,02 µM incrementaron la [Ca++], a 1,48 ñ 0,1 µM. La incubación previa con dantralone bloqueó el efecto del InsP3 sobre [Ca++], en ambos tipos de miofibrillas