C-terminal domain of insulin-like growth factor (IGF) binding protein-6: structure and interaction with IGF-II.

IGFs are important mediators of growth. IGF binding proteins (IGFBPs) 1-6 regulate IGF actions and have IGF-independent actions. The C-terminal domains of IGFBPs contribute to high-affinity IGF binding and modulation of IGF actions and confer some IGF-independent properties, but understanding how they achieve this has been constrained by the lack of a three-dimensional structure. We therefore determined the solution structure of the C-domain of IGFBP-6 using nuclear magnetic resonance (NMR). The domain consists of a thyroglobulin type 1 fold comprising an alpha-helix followed by a loop, a three-stranded antiparallel beta-sheet incorporating a second loop, and finally a disulfide-bonded flexible third loop. The IGF-II binding site on the C-domain was identified by examining NMR spectral changes upon complex formation. It consists of a largely hydrophobic surface patch involving the alpha-helix, the first beta-strand, and the first and second loops. The site was confirmed by mutagenesis of several residues, which resulted in decreased IGF binding affinity. The IGF-II binding site lies adjacent to surfaces likely to be involved in glycosaminoglycan binding of IGFBPs, which might explain their decreased IGF affinity when bound to glycosaminoglycans, and nuclear localization. Our structure provides a framework for understanding the roles of IGFBP C-domains in modulating IGF actions and conferring IGF-independent actions, as well as ultimately for the development of therapeutic IGF inhibitors for diseases including cancer.

Calcitonin receptor isoforms expressed in the developing rat kidney.

BACKGROUND: Development in the metanephric-kidney transition period involves the precise expression of paracrine and autocrine events in an ordered spatio-temporal manner. Expression of these molecular events is tightly controlled and includes positive and negative growth factors and cognate receptors within close proximity in developing structures in the expanding renal cortex and medulla. The expression of calcitonin receptor (CTR) isoforms C1a and C1b in this context has not previously been described. Our current study also explored the relationship between the expression of CTR isoforms and amylin binding sites. METHODS: Techniques included immunohistochemistry with novel antibodies that detect CTR isoforms, real time PCR for the quantification of CTR isoforms, Western blot and in vitro autoradiography, on tissues from embryo day 18 to postnatal day 30. RESULTS: The CTR C1a isoform is expressed in the ureteric ducts of the metanephros and both isoforms are expressed in the developing distal convoluted tubules, ascending limbs of the loop of Henle and collecting ducts in the postnatal rat kidney. There was a 60-fold excess of C1a versus C1b isoforms. An apparent molecular weight of 63 kD was found. In vitro autoradiography demonstrated that while amylin binding sites were predominantly in the cortex, CTR expression was largely localized in the medulla in an earlier event, followed by cortical expression. CONCLUSIONS: CTR C1a protein expression has been identified in the ureteric ducts in the metanephros and both isoforms expressed in the distal portions of the developing nephrons and collecting ducts. Since amylin binding sites have been localized on the proximal tubules of the cortex, it is unlikely that amylin receptors can be represented by modification of CTR affinity with receptor activity modifying proteins in the kidney.