Studies on the Structure and Function of Glycosylated and Nonglycosylated neu Differentiation Factors: SIMILARITIES AND DIFFERENCES OF THE α AND β ISOFORMS

Hsieng S Lu,David Chang,John S Philo,Ke Zhang,Linda O Narhi,Naili Liu,Mei Zhang,Jilin Sun,Jie Wen,Donna Yanagihara,Devarajan Karunagaran,Yosef Yarden,Barry Ratzkin

doi:10.1074/jbc.270.9.4784

Hsieng S Lu, David Chang + Show 11 more

Open Access

https://doi.org/10.1074/jbc.270.9.4784

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Abstract

Comparative analyses of both glycosylated and nonglycosylated neu differentiation factor (NDF) isoforms revealed significant similarities and differences of their overall structures and functions. Biophysical analyses confirmed that all NDF isoforms are monomeric, but have an extended ellipsoidal shape in solution. All full-length NDFs are similar in secondary and tertiary structures and they contain no alpha-helix but are abundant in beta-strand structures. A small NDF fragment containing only the epidermal growth factor domain is also rich in beta-strand structures, but exhibits tertiary structure different from the long NDF forms. Monoclonal antibodies that selectively recognize epidermal growth factor domains of human NDF-alpha and -beta can specifically bind the respective NDF-alpha and -beta isoforms independent of NDF origins. Western blot analysis and quantitative binding assays further identify that an NDF preparation produced naturally from Rat1-EJ cells contains both alpha and beta isoforms in a 3 to 2 ratio. In receptor-binding competition experiments, human and rat NDF-beta isoforms have higher affinity than NDF-alpha isoforms. NDF-beta isoforms can dramatically enhance the stimulation of DNA synthesis for transfected NIH3T3 cells that overexpress HER-3 and HER-4 receptors, while NDF-alpha isoforms can only stimulate proliferation of HER-4-transfected cells with lower activity. Taken together, NDF-alpha and -beta isoforms share similar gross protein conformations but are biologically distinct.

Highlights

Comparative analyses of both glycosylated and nonglycosylated neu differentiation factor (NDF) isoforms revealed significant similarities and differences of their overall structures and functions
Recent studies have shown that NDF or heregulins induce tyrosine phosphorylation ofHER-4 [8] and that NDF isofonns bind to both HER-3 and HER-4 receptors, [9] suggesting that instead of HER-2, HER-3 and HER-4 may function as direct physiological receptors for neuregulin
Gel filtration analysis of E. coliderived, nonglycosylated NDF isoforms gave molecular masses of 53,000-55,000 kDa which is significantly larger than expected. These results suggest that NDFs may be dimeric in solution

Summary

Introduction

Comparative analyses of both glycosylated and nonglycosylated neu differentiation factor (NDF) isoforms revealed significant similarities and differences of their overall structures and functions. Pro-NDFs exist in multiple isoforms which exhibit significant sequence differences in the third disulfide loop of the EGF domain, the juxtamembrane region, and the C-terminal cytoplasmic tail [5]. These isoforms together with two other distinct molecules, glial cell growth fac-. The molecular diversity of neuregulin has raised interesting questions on aspects of structural and functional multiplicity of various isoforms It is still unknown whether each molecular species within the family can display distinct biological actions in its membrane-bound or secreted form.

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