Development of recombinant human prolactin receptor antagonists by molecular mimicry of the phosphorylated hormone

Endocrinology
T J ChenA M Walker

Abstract

Previous studies have demonstrated that naturally phosphorylated PRL antagonizes the growth-promoting effects of unmodified PRL in two different PRL-responsive cell lines. In this study our aim was to produce a molecular mimic of phosphorylated PRL by substituting a fairly bulky, negatively charged amino acid (glutamate or aspartate) for the normally phosphorylated serine [serine 179 in human PRL (hPRL)]. In addition, because of the marked effect of phosphorylation on biological activity, we investigated the importance of the unmodified serine in the growth-promoting activity of PRL. hPRL complementary DNA was obtained from the American Type Culture Collection and subcloned into pT7-SCII after site-directed mutagenesis using the deoxyuridine approach. Proteins were expressed in Escherichia coli BL21 (DE3) and were primarily found in inclusion bodies. Agonist and antagonist activities of each serine 179 mutant were assessed using the Nb2 bioassay. Compared with standard hPRL, the recombinant wild-type was more active in the Nb2 assay, attesting to both the absence, or low level, of endotoxin contamination in preparations from these cells and the appropriate folding of the molecule. The aspartate and glutamate mutants had no intr...Continue Reading

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Citations

Jul 14, 2010·Journal of Biomedicine & Biotechnology·Herbert Rodrigues GoulartCarlos Roberto Jorge Soares
Feb 3, 2007·The Journal of Reproduction and Development·Kotaro HoriguchiToshio Harigaya
Jan 26, 2008·Journal of Mammary Gland Biology and Neoplasia·KuangTzu HuangAmeae M Walker
Oct 20, 2007·Molecular and Cellular Endocrinology·Esther A GuzmánLaurie B Owen
Aug 3, 2007·Molecular and Cellular Endocrinology·Ameae M Walker
Jun 23, 2015·Cancer Letters·Tomohiro YonezawaAmeae M Walker
Oct 30, 2004·Annals of Medicine·Jessica HarrisChristopher J Ormandy
Aug 22, 2003·Protein Science : a Publication of the Protein Society·Scott T R WalshAnthony A Kossiakoff
Feb 5, 2008·Journal of Mammary Gland Biology and Neoplasia·Charles V ClevengerFeng Fang
Apr 9, 2005·Endocrine Reviews·Vincent GoffinPaul A Kelly
Dec 30, 2011·American Journal of Physiology. Endocrinology and Metabolism·Mary Y LorensonAmeae M Walker
Sep 9, 2020·Expert Opinion on Therapeutic Targets·Antonela S AsadMarianela Candolfi
Nov 30, 2000·American Journal of Physiology. Endocrinology and Metabolism·D CossA M Walker
Oct 4, 2000·Physiological Reviews·M E FreemanG Nagy
Dec 3, 2016·Journal of Mammary Gland Biology and Neoplasia·Riva Dill, Ameae M Walker
Oct 4, 2007·American Journal of Physiology. Endocrinology and Metabolism·Valencia L WilliamsAmeae M Walker
Sep 24, 2020·The Journal of Reproduction and Development·Kazunori MorohoshiToshio Harigaya
May 22, 2003·The Journal of Biological Chemistry·Matthew J NaylorChristopher J Ormandy
May 10, 2008·The Journal of Biological Chemistry·L Anders SvenssonJens Breinholt
Jun 26, 2003·The Journal of Biological Chemistry·Sophie BernichteinVincent Goffin

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