Chemical Synthesis, Structural Modeling, and Biological Activity of the Epidermal Growth Factor-like Domain of HumanCripto

Abstract

Cripto, also known as human teratocarcinoma-derived growth factor 1 (TDGF-1), contains a 40 amino acid region with some similarity to the epidermal growth factor (EGF) domain. However, sequence homology is largely restricted to the classical cysteine/glycine motif with only limited similarities in other regions. Significant differences to human EGF include the absence of all seven residues between the two N-terminal half-cystines and a five-residue shorter loop between the third and fourth half-cystines. We examine the hypothesis that, in spite of these differences, cripto can adopt the characteristic EGF-like 1-3, 2-4, 5-6 disulfide bond pattern. A comparative structural model of the growth factor cripto was constructed on the basis of its similarity to EGF, transforming growth factor alpha (TGF-alpha), and the EGF-like domain of human clotting factor IX. The predicted disulfide bridges and disulfide-bridged loops were analyzed and appear viable in the modeled structure. Moreover, to ascertain the importance of disulfide arrangement for cripto bioactivity, two 47-residue peptides were synthesized and then refolded using either a simple oxidative or a controlled sequential refolding protocol. The cripto peptides were tested for their ability to stimulate MAP-kinase activity, for inhibition of beta-casein induction, and for Shc phosphorylation in MDA-MB 453 human mammary carcinoma cells and HC-11 mouse mammary epithelial cells. Data suggest that cripto does adopt the 1-3, 2-4, 5-6 disulfide pattern and thus forms the classical EGF-like fold in spite of the significant deletions within the folding domain. The predicted structure of cripto shows some of the characteristics of both the ErbB1- and ErbB3/ErbB4-binding growth factors.

The EGF-CFC family: novel epidermal growth factor-related proteins in development and cancer.

“…However, recombinant CR-1 in radioreceptor competition assays with 125 I-EGF in human mammary MCF-10A cells or A431 epidermoid carcinoma cells failed to demonstrate any binding to the EGFR on these cells (Kannan et al 1997). Reciprocally, 125 I-CR-1 binding to several different human breast carcinoma cells lines could be blocked by CR-1 but not by EGF, TGFα, BTC, AR or HRGβ1.…”

“…Reciprocally, 125 I-CR-1 binding to several different human breast carcinoma cells lines could be blocked by CR-1 but not by EGF, TGFα, BTC, AR or HRGβ1. Finally, recombinant CR-1 using BAF/3 mouse lymphoid cells that were ectopically expressing the human EGFR, erb B-2, erb B-3, or erb B-4 either alone or in various pairwise combinations was unable to activate directly the tyrosine phosphorylation of these tyrosine kinase receptors, demonstrating that CR-1 was not able to bind directly to these type 1 receptor kinases either as homodimers or as heterodimers (Kannan et al 1997).…”

“…Since the EGF-CFC peptides lack the A loop, this suggests that these proteins probably do not bind directly to any of the known erb B-related tyrosine kinase receptors. Molecular modeling of this modified EGF-like motif in the EGF-CFC proteins has shown a closely packed three-disulfide stacked arrangement with a disulfide β-cross motif for the first and second disulfides (Lohmeyer et al 1997). The EGF-motif contains two β-hairpins of six residues in the NH 2 -and COOH-terminus respectively.…”

“…In addition, there is no source for recombinant nodal protein to ascertain its potential interaction with other EGF-CFC proteins in various mammalian cell types. Nevertheless, recent experiments with mouse mammary HC-11 cells have demonstrated that recombinant CR-1 is able to enhance the tyrosine phosphorylation of the SH2-adapter protein Shc leading to the subsequent downstream activation of the ras/raf/MAPK signaling pathway (Kannan et al 1997). It was shown that exogenous CR-1 protein binds to high affinity, saturable binding site(s) on HC-11 cells as well as on several different human breast cancer cell lines.…”

Molecular Analysis of the Cripto Growth Factor Receptor

“…Transfection of human Cripto results in transformation of and NOG-8 mouse normal mammary epithelial cells [8]. Experiments using the refolded CRIPTO peptide have shown that CRIPTO can induce tyrosine phosphrylation of the SH2 adapter protein She in HC-11 cells, resulting in increased association of She with Grb2 and elevated MAPK kinase activity [2,9]. More recently it has been shown that recombinant CRIPTO can transactivate erbB-A receptor, but not EGF receptor, erbB-2 and erbB-3 [10].…”