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.