Synthesis and properties of an EGF-like domain (residues 361-406) in the extreme N-terminal region of the mouse EGF precursor.
Various proteins contain EGF-like domains that are not ligands for the EGF receptor. In the present study a cognate polypeptide for residues 361-406 of the mouse EGF precursor was synthesized by the solid-phase method. The product was renatured under oxidative conditions since it probably has an EGF-like array of three cystine disulfide bonds in its native state. HPLC analysis of the renaturation reaction revealed formation of a peak material with no apparent free-SH groups. Accordingly, the HPLC retention time of this product was readily increased by treatment (reduction of disulfides) with dithiothreitol. The renatured 46-mer (PEGF-1) did not displace 125I-EGF bound to rat liver membranes and 125I-PEGF-1 did not exhibit specific binding to membrane preparations from the mouse liver, mammary gland, or kidney, with or without Ca2+ in the binding medium. Although PEGF-1 contains a putative Ca2+ binding motif, specific binding of this cation by the polypeptide could not be demonstrated by electromobility shiff or incubation with 45Ca2+. Immunoassay of PEGF-1 and EGF in fractions obtained following gel filtration of mouse urine revealed multiple peaks of PEGF-1 immunoreactivity with the major peaks eluting at an Mr > 30 kDa. In contrast, virtually all the EGF immunoreactivity eluted at a volume similar to that of 125I-EGF. These data suggest that selective cleavage of the PEGF-1 domain from the precursor does not occur with the proclivity known for that of EGF. Instead, the PEGF-1 probably functions coordinately with other EGF-like domains while tethered to the precursor backbone. Finally, localization of PEGF-1 immunoreactivity occurred only in cell populations of the mouse previously demonstrated as sites for EGF/EGF precursor, which suggests that PEGF-1 is exclusively a domain of the EGF precursor.
Diaugustine
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RP
Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC 27709, USA. diaugus2@niehs.nih.gov
Henry
R
R
Sewall
C H
CH
Suarez-Quian
C A
CA
Walker
M P
MP
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002132:Calcium Radioisotopes; D004815:Epidermal Growth Factor / Q000138:chemical synthesis / Q000378:metabolism* / Q000652:urine; D066246:ErbB Receptors / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism / Q000652:urine; D011498:Protein Precursors / Q000138:chemical synthesis / Q000378:metabolism*; D020673:Protein Renaturation; D017434:Protein Structure, Tertiary; D051381:Rats
D002132:Calcium Radioisotopes; D008565:Membrane Proteins; D010446:Peptide Fragments; D011498:Protein Precursors; C042652:epidermal growth factor precursor; D004815:Epidermal Growth Factor; D066246:ErbB Receptors