Search for a Novel Gla-containing Growth Factor as the Ligand for Mer Receptor Tyrosine Kinase

我们之前发现Gas6是Axi， Sky和Mer受体酪氨酸激酶的共同配体。定量结合分析表明，Gas6与Mer的结合亲和力较低，Kd值为29.0 nM。因此，我们假设存在gas6相关蛋白作为Mer受体的配体。在这项研究中，我们寻找可能作为Mer首选配体的gas6相关蛋白。Gas6的结构类似于蛋白S，由一个Gla结构域、四个egf样结构域和一个c端性激素结合球蛋白（SHBG）样结构域组成。当研究每个结构域在Gas6的受体结合和生物活性中的作用时，我们发现通过抑制Gla结构域的γ -羧化，受体结合和有丝分裂活性显著降低，而仅由shbg样结构域组成的Gas6突变体保留了这两种活性。因此，shbg样结构域显然是Gas6活性不可缺少的实体，Gla结构域的γ -羧化在保持天然Gas6活性方面具有调节作用。我们利用PCR和低严格杂交技术寻找编码gas6相关蛋白的cDNA克隆，但未能克隆编码该蛋白。雄激素结合蛋白与Gas6的SHBG-like结构域序列相似性较低，对Mer、Sky和Axl受体没有结合能力。因此，我们没有检测到除s蛋白外的任何与gas6相关的蛋白。可能在生物体中，gas6诱导Mer的激活需要辅因子或辅受体。Axl-Fc是AxI受体的一种可溶性形式，由AxI的胞外结构域和免疫球蛋白的Fc区组成，对血管平滑肌细胞上Gas6的促生长活性具有抑制作用。

We previously identified that Gas6 is a common ligand for Axi, Sky, and Mer receptor tyrosine kinases. Quantitative binding analysis revealed that the binding affinity of Gas6 to Mer is relatively low, with a Kd value of 29.0 nM.Thus, we assumed the existence of the Gas6-related protein as a ligand for Mer receptor. In this study, we searched for the Gas6-related protein that may function as the preferable ligand for Mer. Gas6 has a structure similar to that of protein S and is composed of a Gla domain, four EGF-like domains and a C-terminal sex hormone-binding globulin (SHBG)- like domain. When examining the role of each domain in receptor-binding and biological activities of Gas6, we found that receptor-binding and mitogenic activities were markedly reduced by inhibiting gamma -carboxylation of the Gla domain, while a Gas6 mutant composed of only an SHBG-like domain retained both of these activities. Thus, the SHBG-like domain is apparently an entity indispensable for Gas6 activities, and gamma -carboxylation of the Gla domain has a regulatory role in retaining the activity of native Gas6. We searched for the cDNA clones coding for Gas6-related proteins, using PCR and low stringency hybridization techniques, but we failed to clone coding, for such protein. Androgen-binding protein, which has low sequence similarity to the SHBG-like domain of Gas6, showed no binding ability to Mer, Sky or Axl receptor. Thus, we have not detected any Gas6-related protein other than protein S.It could be that Gas6-induced activation of Mer require co-factors or co-receptors in living organisms. We also showed that Axl-Fc, a soluble form of AxI receptor composed of the extracellular domain of AxI and the Fc region of immunoglobulin, had an inhibitory function for the growth potentiating activity of Gas6 on vascular smooth muscle cells.