Headey Stephen J, Leeding Kerri S, Norton Raymond S, Bach Leon A
University of Melbourne, Department of Medicine, Austin Hospital, Heidelberg 3084, Australia.
J Mol Endocrinol. 2004 Oct;33(2):377-86. doi: 10.1677/jme.1.01547.
Insulin-like growth factors IGF-I and IGF -II are important mediators of growth. A family of six high affinity IGF binding proteins (IGFBPs) modulate IGF action. IGFBPs have three domains, of which the N- and C-domains are involved in high affinity IGF binding. IGFBP-6 is unique in its 20-100-fold IGF-II binding specificity over IGF-I. The aim of this study was to determine the contributions of the N- and C-domains of IGFBP-6 to its IGF binding properties. We confirmed that differential dissociation kinetics are responsible for the IGF-II binding preference of IGFBP-6. The N-domain has rapid association kinetics, similar to full-length IGFBP-6, but both IGF-I and -II dissociate rapidly from this domain, thereby reducing its binding affinity for IGF-II approximately 50-fold. However, the N-domain binds IGF-I and -II with similar affinities and it has a similar IGF-I binding affinity to full-length IGFBP-6. This suggests that the C-domain confers the IGF-II binding preference of IGFBP-6; indeed, IGF-I bound inconsistently with very low affinity to the C-domain. Coincubation studies showed that isolated N- and C-domains of IGFBP-6 do not strongly cooperate to enhance IGF binding. The results of the binding studies are supported by the effects of the IGFBP-6 domains on IGF-induced colon cancer cell proliferation; the N-domain inhibited IGF-II induced proliferation with approximately 20-fold lower potency than IGFBP-6 and it was equipotent in inhibiting IGF-I- and IGF-II-induced proliferation. Coincubation of C-domain had no additional effect on N-domain-induced inhibition of proliferation. In conclusion, both the N- and C-domains of IGFBP-6 are involved in IGF binding, the C-domain is responsible for the IGF-II binding preference of IGFBP-6 and intact IGFBP-6 is necessary for high affinity IGF binding.
胰岛素样生长因子IGF-I和IGF-II是生长的重要调节因子。一个由六种高亲和力IGF结合蛋白（IGFBPs）组成的家族调节IGF的作用。IGFBPs有三个结构域，其中N端和C端结构域参与高亲和力的IGF结合。IGFBP-6对IGF-II的结合特异性是对IGF-I的20至100倍，这一点很独特。本研究的目的是确定IGFBP-6的N端和C端结构域对其IGF结合特性的贡献。我们证实，不同的解离动力学是IGFBP-6对IGF-II结合偏好的原因。N端结构域具有快速的结合动力学，类似于全长IGFBP-6，但IGF-I和IGF-II都能从该结构域迅速解离，从而使其对IGF-II的结合亲和力降低约50倍。然而，N端结构域对IGF-I和IGF-II的结合亲和力相似，并且它对IGF-I的结合亲和力与全长IGFBP-6相似。这表明C端结构域赋予了IGFBP-6对IGF-II的结合偏好；实际上，IGF-I与C端结构域的结合不一致，亲和力非常低。共孵育研究表明，IGFBP-6分离的N端和C端结构域不会强烈协同增强IGF结合。结合研究的结果得到了IGFBP-6结构域对IGF诱导的结肠癌细胞增殖影响的支持；N端结构域抑制IGF-II诱导的增殖，其效力比IGFBP-6低约20倍，并且在抑制IGF-I和IGF-II诱导的增殖方面效力相当。C端结构域的共孵育对N端结构域诱导的增殖抑制没有额外影响。总之，IGFBP-6的N端和C端结构域都参与IGF结合，C端结构域是IGFBP-6对IGF-II结合偏好的原因，完整的IGFBP-6是高亲和力IGF结合所必需的。
