Demonstration of multiforms of insect cytokine receptors

生长阻滞肽(GBP)是昆虫细胞因子家族的一员。GBP是一个由25个氨基酸残基组成的小肽，但它具有多种功能，包括抑制幼虫生长、细胞增殖、麻痹诱导、心脏加速、免疫细胞刺激和形态发生。本研究合成了GBP的缺失类似物，以研究GBP的结构与促有丝分裂和浆细胞(血细胞的一种)扩散等生物活性的关系。结果表明，具有促有丝分裂活性的GBP的最小结构为2~23 GBP，而具有浆细胞铺展活性的GBP的最小结构为1~22 GBP，提示GBP的多功能性可能是由不同形式的GBP受体介导的。为了证明这一推测，我们通过竞争性受体结合试验分析了不同类型细胞中的GBP受体。结果表明，高五细胞含有高亲和力(K_d=0.26)和低亲和力(K_d=13.7)两种类型的GBP受体，而血细胞只有一种中等亲和力的GBP受体(K_d=1.26)。这些结果表明，至少在昆虫培养细胞和血细胞中，GBP受体具有异质性。此外，我们在血细胞中确定了两种不同类型的GBP受体候选者，大约60 kDa和77 kDa蛋白。前者除了一个跨膜区外，不包含任何不同的结构域。后者含有一个跨膜区，外细胞区和内细胞区分别含有ITAM(基于免疫受体酪氨酸的激活基序)和Ig G样结构域。我们进一步发现，该分子胞质区域的酪氨酸残基被GBP磷酸化。我们现在正试图证明GBP是否与这些细胞膜蛋白特异性结合。

Growth-blocking peptide (GBP) is a member of an insect cytokine family. GBP is a small peptide consists of 25 amino acid residues, but it exerts diverse functions including larval growth retardation, cell proliferation, paralysis induction, cardioacceleration, immune cell stimulation, and morphogenesis. In this study, deletion analogs of GBP were synthesized to investigate the relationship between the structure of GBP and the biological activities such as mitogenic and plasmatocyte (a type of hemocytes) spreading activities. The results indicated that the minimal structure of GBP containing mitogenic activity is 2-23 GBP, whereas that with plasmatocyte spreading activity is 1-22 GBP, indicating the possibility that multifunctional properties of this peptide may be mediated by different forms of GBP receptors. In order to demonstrate this speculation, we analyzed GBP receptors in different types of cells by a competitive receptor binding assay with ^<125>I-GBP. The results showed that High Five cells contain two types of GBP receptors : high affinity (K_d=0.26) and low affinity (K_d=13.7), while hemocytes contain a single class of GBP receptor with a medium affinity (K_d=1.26). These results demonstrated a heterogeneity of GBP receptors at least in insect culture cells and hemocyte cells. Further, we identified two different types of GBP receptor candidates in hemocytes, approximately 60 kDa and 77 kDa proteins. The former protein does not contain any distinct domain structures other than one transmembrane domain. However, the latter protein contains one transmembrane domain, and ITAM (immunoreceptor tyrosine-based activation motif) and IgG like domains in the outer cell region and inner cell region, respectively. We further found that tyrosine residues in the cytoplasmic region of this molecule are phosphorylated by GBP. We are now trying to demonstrate whether GBP specifically binds to these cell membrane proteins.