Eder M, Ernst T J, Ganser A, Jubinsky P T, Inhorn R, Hoelzer D, Griffin J D
Department of Hematology, University of Frankfurt, Federal Republic of Germany.
J Biol Chem. 1994 Dec 2;269(48):30173-80.
The high affinity receptor for granulocyte-macrophage colony-stimulating factor (GM-CSF) is composed of at least two subunits, an 85-kDa low affinity GM-CSF-binding protein (alpha-GMR) and a 120-kDa beta-subunit (beta-GMR) necessary for high affinity binding and signal transduction. Previous studies have shown that deletion of the intracellular domain of alpha-GMR inactivates the receptor's ability to support proliferation, but has no effect on GM-CSF binding. Using anti-alpha-GMR- and anti-beta-GMR-specific antibodies, we show that alpha-GMR and beta-GMR coprecipitate only after GM-CSF binding, suggesting that binding of GM-CSF induces stabilization or assembly of an activated receptor complex involving recruitment of beta-GMR chains. To understand the contribution of each subunit of this receptor to the generation of an activated receptor complex, we attempted to construct minimal receptors with some or all of the functions of the wild-type heterodimer. We found that a hybrid human alpha/beta-GMR molecule in which the extracellular and transmembrane segments are composed of alpha-GMR sequences and the intracellular segment is composed of beta-GMR bound GM-CSF with low affinity, but activated tyrosine kinase activity, induced receptor internalization, and supported short- and long-term proliferation of transfected Ba/F3 cells. At least 1 ng/ml human GM-CSF was required for growth stimulation, and maximal proliferation occurred at a concentration of 10 ng/ml. This was 10-100-fold more than needed to stimulate growth of Ba/F3 cells expressing both full-length human alpha-GMR and beta-GMR and 1000-fold less than needed to stimulate growth of Ba/F3 cells expressing only human alpha-GMR. These results indicate that the cytoplasmic domain of alpha-GMR is not required to initiate a unique signaling event for proliferation in Ba/F3 cells, but can be functionally replaced by the cytoplasmic domain of beta-GMR.
粒细胞-巨噬细胞集落刺激因子（GM-CSF）的高亲和力受体由至少两个亚基组成，一个85 kDa的低亲和力GM-CSF结合蛋白（α-GMR）和一个120 kDa的β亚基（β-GMR），后者是高亲和力结合和信号转导所必需的。先前的研究表明，缺失α-GMR的胞内结构域会使受体支持增殖的能力失活，但对GM-CSF结合没有影响。使用抗α-GMR和抗β-GMR特异性抗体，我们发现α-GMR和β-GMR仅在GM-CSF结合后才会共沉淀，这表明GM-CSF的结合诱导了一个活化受体复合物的稳定或组装，涉及β-GMR链的募集。为了了解该受体的每个亚基对活化受体复合物生成的贡献，我们试图构建具有野生型异二聚体部分或全部功能的最小受体。我们发现，一种杂交的人α/β-GMR分子，其胞外和跨膜区段由α-GMR序列组成，胞内区段由β-GMR组成，该分子以低亲和力结合GM-CSF，但激活酪氨酸激酶活性，诱导受体内化，并支持转染的Ba/F3细胞的短期和长期增殖。生长刺激至少需要1 ng/ml的人GM-CSF，最大增殖发生在10 ng/ml的浓度下。这比刺激同时表达全长人α-GMR和β-GMR的Ba/F3细胞生长所需的浓度高10 - 100倍，比刺激仅表达人α-GMR的Ba/F3细胞生长所需的浓度低1000倍。这些结果表明，α-GMR的胞质结构域对于在Ba/F3细胞中启动增殖的独特信号事件不是必需的，但可以在功能上被β-GMR的胞质结构域替代。
