Novel Recombinant Analogues of Bovine Placental Lactogen

Abstract

Two new analogues of bovine placental lactogen (bPL), bPL(G133K) and bPL(G133R), were expressed in Escherichia coli, refolded, and purified to a native form. Binding experiments, which are likely to represent the binding to site 1 only, to intact FDC-P1 cells transfected with rabbit (rb) growth hormone receptor (GHR) or with human (h) GHR, to Nb2 rat lymphoma cells, or to rabbit mammary gland membranes prolactin receptor (PRLR), revealed only small or no reduction in binding capacity. The complex formation between these analogues and receptor extracellular domains (R-ECD) of various hormones was determined by gel filtration. Wild type bPL yielded 1:2 complex with hGHR-ECD, rat PRLR-ECD, and rbPRLR-ECD, whereas both analogues formed only 1:1 complexes with all R-ECDs tested. Real time kinetics experiments demonstrated that the ability of the analogues to form homodimeric complexes was compromised in both PRLR-and GHR-ECDs. The biological activity transduced through lactogenic receptors in in vitro bioassays in rabbit mammary gland acini culture and in Nb2 cells was almost fully retained, whereas the activity transduced through somatogenic receptors in FDC-P1 cells transfected with rbGHRs or with hGHRs was abolished. Both analogues exhibited antagonistic activity in the latter cells. To explain the discrepancy between the effect of the mutation on the signal transduced by PLR versus GHRs we suggest that: 1) the mutation impairs the ability of site 2 of bPL to form a stable homodimeric complex with both lactogenic and somatogenic receptors by a drastic shortening of the half-life of 2:1 complex; 2) the transient existence of the homodimeric complex is still sufficient to initiate the signal transduced through lactogenic receptors but not through somatogenic receptors; and 3) one possible reason for this difference is that JAK2, which serves as a mediator of both receptors, is already associated with lactogenic receptors prior to hormone binding-induced receptor dimerization, whereas in somatogenic receptors the JAK2 receptor association occurs subsequently to receptor dimerization.

Functional Heterodimerization of Prolactin and Growth Hormone Receptors by Ovine Placental Lactogen

“…In the case of oGHR-ECD, which at that concentration failed to bind over 2,700 RU (stoichiometry, 1:1), the dissociation kinetics clearly showed a good fit to a one-site interaction model, and the calculated k off value (mean Ϯ S.D., n ϭ 3) was 3.4 Ϯ 0.30 ϫ 10 Ϫ4 min Ϫ1 . In contrast, although bPRLR reached a maximum of about 2,200 RU, the dissociation kinetics was indicative of a two-site interaction model with loose binding at each site, which, as shown by us previously is characteristic of homologous interaction with PRLRs (18,19,29). The two dissociation constants (mean Ϯ S.D., n ϭ 3 or 4) were, respectively, k1 off ϭ 3.08 Ϯ 0.33 ϫ 10 Ϫ3 min Ϫ1 and k2 off ϭ 4.9 Ϯ 0.15 ϫ 10 Ϫ2 min…”

“…Comparative binding studies of oPL and oGH to fetal liver microsomes along with the demonstration of oGHR mRNA in fetal liver prompted several research groups to suggest that oGH and oPL bind to identical or at least related, proteins (13)(14)(15). Using a similar approach, we previously studied the biological activity of the three ruminant PLs in several in vitro bioassays, in which the signal was transduced through heterologous (mouse, rabbit, and human) GHRs (9,11,(15)(16)(17)(18)(19). In all cases, the activity of bPL, oPL, or caprine PL was equal or similar to that of oGH or bGH and in the case of hGH receptors, also to hGH.…”

Biological Properties of Human Prolactin Analogs Depend Not Only on Global Hormone Affinity, but Also on the Relative Affinities of Both Receptor Binding Sites

“…It has been hypothesized (46) that such a difference between the two receptors was due to the fact that the receptor-associated tyrosine kinase JAK2 (47) is constitutively bound to the PRLR (48) but not to the GHR (49). Hence, very transient formation of PRLR dimers might be sufficient to trigger signaling cascades, whereas in the case of the GHR, formation of more stable dimers might be required to recruit JAK2 prior to engagement of signaling molecules (46). Although this hypothesis is attractive, it awaits further demonstration.…”

“…Although the agonistic activity of this analog is almost completely abolished, its antagonistic properties are relatively low due to its reduced affinity. If the hypothesis of Helman and colleagues is correct (46), the design of more potent hPRL antagonists should rather focus on a total inhibition of binding site 2 than on increasing site 1 affinity. Indeed, as demonstrated by the partial agonistic activity of the D183E/ G129R analog, the G129R mutation is not as potent as initially believed for definitely blocking binding site 2 (24).…”

Ruminant Placental Lactogens Act as Antagonists to Homologous Growth Hormone Receptors and as Agonists to Human or Rabbit Growth Hormone Receptors

“…Binding studies (Fig. 2) along with the results of Gluckman's group (16) and ours (7,9,21,22) suggest that oPL, cPL, and bPL bind with high affinity to membrane-embedded somatogenic receptors and to GHRECDs. We conclude that this binding occurs through oPL's site I only.…”