The addition of nine residues at the C‐terminus of human prolactin drastically alters its biological properties

Abstract

We have added nine extra residues to the C-terminal of human prolactin and analysed the effect of this mutation on the ability of the hormone to bind to its lactogenic receptor and to induce Nb2 cell division. Both properties are markedly affected when compared to the natural 23-kDa human prolactin. Since no alteration of the global protein folding was detected either by circular dichroism or by infrared spectroscopy, the decrease in biological potency can be exclusively attributed to an effect of the nine additional residues on their near environment. From infrared analysis and secondary structure prediction, the elongated tail is assumed to be involved in a B-sheet with a few residues initially belonging to the fourth helix. Moreover, from the X-ray structures of porcine and human growth hormones, two proteins homologous to prolactins, the nine extra residues are likely to fold within a concave pocket delimited by helices 1 and 4, and the second half of the loop connecting helices 1 and 2 (loop 1). Thereby, we suggest that the additional residues prevent some residues belonging to this pocket from interacting with the lactogenic receptor. This is in perfect agreement with our earlier proposal that the binding site of prolactin to the lactogenic receptor is homologous to that of growth hormone to the somatogenic receptor, i.e. essentially composed of residues belonging to this concave pocket.

S179D-Human PRL, a Pseudophosphorylated Human PRL Analog, Is an Agonist and Not an Antagonist

“…As point mutations can affect regular secondary structures and/or global protein folding, we assessed the ␣-helical content of S179D-hPRL by circular dichroism as previously reported for other hPRL analogs (31,33,40). The profile obtained for S179D-hPRL was almost identical to that obtained with WT hPRL (Fig.…”

Improving the pharmacokinetics/pharmacodynamics of prolactin, GH, and their antagonists by fusion to a synthetic albumin-binding peptide

“…Although the fusion of a small peptide to the carboxylterminus of these ligands was detrimental to their activities in vitro, consistent with the observations of others (Goffin et al 1993), a more favorable PK profile could compensate for their reduced potency, so we examined the PKs of both SA20-hPRL and hPRL-SA20 in mice. The PK behavior of SA20-hPRL and hPRL-SA20 was very similar, indicating that SA20 retains its ability to interact with serum albumin when fused to the amino-or carboxyl-terminus.…”

Characterization of Lactogen Receptor-binding Site 1 of Human Prolactin

“…Binding of hPRL mutants to the lactogen receptor was performed as reported earlier (21,26,28). Briefly, homogenates from 3 ϫ 10 6 Nb2 cells were incubated for 16 h at 25°C with 30,000 -50,000 cpm 125 IhPRL in the presence of increasing amounts of unlabeled native hPRL or hPRL analog (final reaction volume of 0.5 ml).…”

“…Circular Dichroism-hPRL has an ␣-helix content of 45 Ϯ 5% (21,26,28) as determined by circular dichroism. In agreement with the spectrum of native hPRL, all mutants produced in this study displayed the typical curve of all ␣-proteins, with two minima at 222 and 208 nm and a maximum at 195 nm.…”

“…Nb2 cells contain ϳ12,000 PRL receptors/cell (43). As described earlier (21,26,28), Nb2 cells were also used in binding assays of hPRL mutants. Typical Nb2 cell proliferation and binding curves are represented in Fig.…”