Ma Y H, Reusch H P, Wilson E, Escobedo J A, Fantl W J, Williams L T, Ives H E
Cardiovascular Research Institute, University of California, San Francisco 04143.
J Biol Chem. 1994 Dec 2;269(48):30734-9.
The effect of site-specific mutations in the mouse platelet-derived growth factor (PDGF) beta-receptor on activation of the Na+/H+ exchanger was examined in normal murine mammary gland epithelial (NMuMG) and Chinese hamster ovary (CHO) cells. These cells, which do not normally express PDGF receptors, were stably transfected with PDGF beta-receptor cDNA. Intracellular pH and Ca2+ were monitored using fluorescent probes. In both NMuMG and CHO cells expressing wild-type PDGF beta-receptors, PDGF B/B activated the amiloride-sensitive Na+/H+ exchanger. In both cell types, cell alkalinization was reduced by approximately 50% with a receptor mutant Y708F,Y719F which cannot bind phosphatidylinositol (PI) 3'-kinase. An inhibitor of PI 3'-kinase, LY294002, also inhibited alkalinization by 43% in cells with wild-type, but not Y708F,Y719F receptors. PDGF-induced intracellular Ca2+ release was not affected by this mutation. Both alkalinization and Ca2+ release were reduced by nearly 100% with the mutant Y977F,Y989F, which cannot bind phospholipase C gamma (PLC gamma). Y739F, a mutant that fails to bind the GTPase-activating protein did not affect PDGF-induced alkalinization. In protein kinase C (PKC) down-regulated NMuMG cells (wild-type receptor), PDGF no longer activated the Na+/H+ exchanger. In contrast, in PKC down-regulated CHO cells (wild-type receptor), PDGF-induced alkalinization was attenuated by only 37%. This residual activity was unaffected by the Y708F,Y719F mutation, but was completely eliminated by removal of medium Ca2+. These findings indicate that phospholipase C gamma is essential for activation of Na+/H+ exchange. PI 3'-kinase participates in PKC-dependent activation of Na+/H+ exchange by PDGF. In CHO cells, there is a second, Ca(2+)-dependent mechanism for activation of the exchanger.
研究了小鼠血小板衍生生长因子（PDGF）β受体位点特异性突变对正常小鼠乳腺上皮（NMuMG）细胞和中国仓鼠卵巢（CHO）细胞中Na⁺/H⁺交换体激活的影响。这些通常不表达PDGF受体的细胞被稳定转染了PDGFβ受体cDNA。使用荧光探针监测细胞内pH值和Ca²⁺。在表达野生型PDGFβ受体的NMuMG细胞和CHO细胞中，PDGF B/B均激活了阿米洛利敏感的Na⁺/H⁺交换体。在这两种细胞类型中，一种不能结合磷脂酰肌醇（PI）3'-激酶的受体突变体Y708F、Y719F使细胞碱化降低了约50%。PI 3'-激酶抑制剂LY294002在具有野生型受体而非Y708F、Y719F受体的细胞中也使碱化降低了43%。PDGF诱导的细胞内Ca²⁺释放不受此突变影响。不能结合磷脂酶Cγ（PLCγ）的突变体Y977F、Y989F使碱化和Ca²⁺释放均降低了近100%。不能结合GTP酶激活蛋白的突变体Y739F不影响PDGF诱导的碱化。在蛋白激酶C（PKC）下调的NMuMG细胞（野生型受体）中，PDGF不再激活Na⁺/H⁺交换体。相反，在PKC下调的CHO细胞（野生型受体）中，PDGF诱导的碱化仅减弱了37%。这种残余活性不受Y708F、Y719F突变影响，但通过去除培养基中的Ca²⁺可完全消除。这些发现表明磷脂酶Cγ对于Na⁺/H⁺交换体的激活至关重要。PI 3'-激酶通过PDGF参与PKC依赖的Na⁺/H⁺交换体激活。在CHO细胞中，存在第二种Ca²⁺依赖的交换体激活机制。
