Demonstration of multiforms of insect cytokine receptors

生长阻断肽（GBP）是昆虫细胞因子家族的一员。GBP是一种由25个氨基酸残基组成的小肽，但它具有多种功能，包括延缓幼虫生长、细胞增殖、诱导麻痹、加速心脏、刺激免疫细胞和形态发生。本研究合成了GBP的缺失类似物，以研究GBP的结构与生物活性如有丝分裂和浆细胞（一种血细胞）扩散活性之间的关系。结果表明，含有有丝分裂活性的GBP的最小结构为2-23 GBP，而具有浆细胞扩散活性的GBP的最小结构为1-22 GBP，表明该肽的多功能特性可能是由不同形式的GBP受体介导的。为了证明这一推测，我们用^<125>I-GBP的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，High Five细胞含有两种类型的GBP受体：高亲和力（K_d=0.26）和低亲和力（K_d=13.7），而血细胞只含有一类中等亲和力的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受体存在异质性。此外，我们在血细胞中鉴定了两种不同类型的GBP受体候选物，大约60 kDa和77 kDa蛋白。前一种蛋白质除了一个跨膜结构域外不包含任何不同的结构域结构。然而，后者含有一个跨膜结构域，在细胞外区和细胞内区分别含有ITAM（免疫受体酪氨酸激活基序）和IgG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是否特异性地与这些细胞膜蛋白结合。

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.