Gagnon A W, Manning D R, Catani L, Gewirtz A, Poncz M, Brass L F
Department of Medicine, University of Pennsylvania, Philadelphia 19104.
Blood. 1991 Sep 1;78(5):1247-53.
G proteins mediate the interaction between cell surface receptors and intracellular effectors. Recent studies have shown that human retina and rat brain contain mRNA encoding a novel 40-Kd G protein alpha subunit referred to as Gz alpha. Studies with an antiserum selective for the predicted sequence of this protein have suggested that a similar protein is present in human platelets and is phosphorylated during platelet activation. To better understand the structure and function of this protein, the present studies examine its sequence in platelets and compare its abundance in human platelets, megakaryocytes, and two megakaryoblastic cell lines, HEL cells and Dami cells. Three different Gz alpha-selective antisera reacted with a 40-Kd protein in platelet membranes. None of these detected a corresponding protein in HEL or Dami cells, despite the presence in both cell lines of proteins recognized by antisera selective for three members of the Gi alpha family. Northern blotting with a Gz alpha-specific probe prepared from retinal Gz alpha showed two hybridizing species in platelet RNA: a major band at 3.5 kb and a minor band at 2.2 kb. Both were detectable in HEL and Dami cells, but at greatly reduced levels compared with platelets. RNA encoding Gz alpha was also detected in individual human megakaryocytes by in situ hybridization. The amount present approached that of Gi alpha 2' the most abundant of the Gi alpha species present in platelets. The complete sequence of the platelet homolog to Gz alpha was determined from platelet RNA amplified by the polymerase chain reaction. The encoded protein was the same as those obtained in brain and retina. Thus, based on immunoreactivity and nucleotide sequencing, platelets and megakaryocytes contain substantial quantities of a protein identical to brain and retinal Gz alpha. The paucity of Gz alpha protein and RNA in the megakaryoblastic cell lines suggests that either there has been a selective loss of the ability to synthesize Gz alpha from these cells or that Gz alpha appears at a later stage in megakaryocyte development than does Gi alpha.
G蛋白介导细胞表面受体与细胞内效应器之间的相互作用。最近的研究表明，人类视网膜和大鼠大脑含有编码一种新型40 kDa G蛋白α亚基（称为Gzα）的mRNA。用对该蛋白预测序列具有选择性的抗血清进行的研究表明，人血小板中存在类似的蛋白，并且在血小板活化过程中会发生磷酸化。为了更好地理解该蛋白的结构和功能，本研究检测了其在血小板中的序列，并比较了其在人血小板、巨核细胞以及两种巨核母细胞系（HEL细胞和Dami细胞）中的丰度。三种不同的Gzα选择性抗血清与血小板膜中的一种40 kDa蛋白发生反应。尽管这两种细胞系中存在被针对Giα家族三个成员的抗血清识别的蛋白，但这些抗血清均未在HEL或Dami细胞中检测到相应的蛋白。用从视网膜Gzα制备的Gzα特异性探针进行Northern印迹分析，在血小板RNA中显示出两个杂交条带：一条主要条带在3.5 kb处，一条次要条带在2.2 kb处。两者在HEL和Dami细胞中均能检测到，但与血小板相比水平大大降低。通过原位杂交在单个人类巨核细胞中也检测到了编码Gzα的RNA。其含量接近Giα2（血小板中存在的Giα种类中最丰富的）的含量。通过聚合酶链反应扩增血小板RNA，确定了与Gzα同源的血小板蛋白的完整序列。编码的蛋白与在大脑和视网膜中获得的蛋白相同。因此，基于免疫反应性和核苷酸测序，血小板和巨核细胞含有大量与大脑和视网膜Gzα相同的蛋白。巨核母细胞系中Gzα蛋白和RNA的缺乏表明，要么这些细胞合成Gzα的能力发生了选择性丧失，要么Gzα在巨核细胞发育中的出现阶段比Giα更晚。
