| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1993: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1992: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
The ubiquitous plasma membrane Na^+/H^+ exchanger (NHE1 isoform) is activated in response to various growth factors and mechanical signals such as osmotic changeand cell spreading, leading to a sustained increase in cytosolic pH (pHi). This activation results from the alkaline shift in pHi-sensitivity of the intracellular "H+-modifier" site. To examine the intracellular Ca2+ (Ca2+i) involvement in this process, we studied direct interaction of a ubiquitous Ca2+-dependent regulatory protein, calmodulin (CaM) with NHE1. We found that i) human NHE1 is a novel member of Ca2+/CaM-binding proteins, and ii) NHE1 possesses two CaM-binding sites, A region (residues 636-656) with high affinity for CaM (Kd=-20nM) and B region (residues 656-691) with intermediate affinity (Kd+-350nM). To assess the physiological involvement of CaM-binding sites, several deletion and point mutant cDNAs were generated and CaM-binding-defective mutant exchangers were expressed inthe fibroblastic cells. Mutations of A region, i) icreased pHi-sensitivity and led the exchanger to the partially activated state, ii) abolished Ca2+i-induced activation of NHE1 caused by Ca2+i ionophore (ionomycin), and iii) reduced by 50% or 80% the extent of cytoplasmic alkalinization in response to growth factors (thrombin etc.) or osmotic stress, respectively. These data support the notion that CaM-binding region A functions as an autoinhibitory domain and the exchanger can be activated by attenuating the inhibitory effect through Ca2+/CaM-binding to this region. The data also suggest that this mechanism is partly involved in the activation of NHE1 in response to various external stimuli, and CaM is oneof importatn "signal transducers" which transmit distinct extracellular signals to the "pHi-sensor" of NHE1.
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