Selective Modification of Recombinant Bovine Placental Lactogen by Site-directed Mutagenesis at Its C Terminus

Vashdi-Elberg, Dorit; Staten, Nicholas R.; Sakal, Edna; McKinnie, Russell E.; Djiane, Jean; Krivi, Gwen G.; Gertler, Arieh

doi:10.1074/jbc.271.10.5558

Cited by 12 publications

(6 citation statements)

References 49 publications

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“…However, bPL and oPL (2) are unusual in that they are capable of recognizing and subsequently exhibiting their biological activity through both lactogenic and somatogenic receptors. It has been shown by us (3) and others that bPL is indeed capable of acting through PRLRs in bovine mammary glands (4), in the rat Nb2 lymphoma cell line (5), and through somatogenic receptors in 3T3-F442A preadipocytes (6) and rat hepatocytes (7). We have also shown that these two activities may be selectively modified.…”

supporting

confidence: 56%

“…pressure liquid chromatography gel filtration on a Superdex75 HR 10/30 column was performed with 200-l aliquots of Q-Sepharose column eluted fractions, freeze-dried samples dissolved in H 2 O, or complexes between the soluble recombinant GHR-or PRLR-ECDs and bPL or bPL analogues, using methods described previously (7).…”

Section: Determination Of Monomer Content and Complex Formation-highmentioning

confidence: 99%

“…We have also shown that these two activities may be selectively modified. For instance, in bPL(T188F) or bPL(K73D/F/A), the binding to full-size somatogenic receptors or their ECDs and to bPLR in the endometrium, as well as somatogenic receptor-mediated biological activities, were reduced or abolished, whereas binding to lactogenic receptors or their ECDs and subsequent biological activity was either fully or almost fully retained (7,8). More recently, we have mutated Lys 73 , which corresponds to Arg 64 in hGH and has been identified to be important in the interaction with hGHR-ECD but not with hPRLR-ECD (9).…”

mentioning

confidence: 99%

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Novel Recombinant Analogues of Bovine Placental Lactogen

Helman

Staten

Grosclaude

et al. 1998

Journal of Biological Chemistry

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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.

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supporting

confidence: 56%

Section: Determination Of Monomer Content and Complex Formation-highmentioning

confidence: 99%

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confidence: 99%

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Novel Recombinant Analogues of Bovine Placental Lactogen

Helman

Staten

Grosclaude

et al. 1998

Journal of Biological Chemistry

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“…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.…”

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confidence: 99%

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

Herman¹,

Bignon²,

Daniel³

et al. 2000

Journal of Biological Chemistry

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Although homo-or heterodimerization are common mechanisms for activation of cytokine receptors, crosstalk between two distinct receptors in this superfamily has been never shown. Here we show a physiologically relevant example indicating that such an interaction does occurs, thus raising the hypothesis that heterodimerization between distinct cytokine receptors may be a novel mechanism contributing to the diversity of cytokine signaling. These findings were documented using both surface plasmon resonance and gel filtration experiments and show that ovine placental lactogen (PL) heterodimerizes the extracellular domains (ECDs) of ruminant growth hormone receptor (GHR) and prolactin receptor (PRLR). We also show that PL or PL analogues that exhibit little or no activity in cells transfected with PRLRs and no activity in cells transfected with ovine GHRs exhibit largely enhanced activity in cells cotransfected with both PRLRs and GHRs. Furthermore, chimeric receptors consisting of cytosolic and transmembrane part of ovine GHR or ovine PRLR and ECDs of human granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) ␣ or ␤ were constructed. Upon transfection into Chinese hamster ovary cells along with reporter luciferase gene and stimulation by GM-CSF, a significant increase in luciferase activity occurred when GM-CSFR-␣-PRLR and GM-CSFR-␤-GHR or GM-CSFR-␣-GHR and GM-CSRR-␤-PRLR were cotransfected. In conclusion, we show that ovine PL is capable of functional heterodimerization of GHR and PRLR and that when their cytosolic parts, coupled to the ECD of GM-CSF receptors, are heterodimerized by GM-CSF, they are capable of transducing biological signal.

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“…Recombinant bPL, bPL G133R, bPL K73D, bPL T188D, oPL, oPL T185D, oPL G130R, hGH and nonglycosylated recombinant rat (r) and bovine PRLR-ECDs were prepared as described previously (Sakal et al 1997, Krivi et al 1989, Vashdi-Elberg et al 1996. Carrier-free 125 I-labelled Na was purchased from New England Nuclear Corp. (Boston, MA, USA).…”

Section: Methodsmentioning

confidence: 99%

Mutations of ovine and bovine placental lactogens change, in different ways, the biological activity mediated through homologous and heterologous lactogenic receptors

Helman

Herman²,

Paly³

et al. 2001

Journal of Endocrinology

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The biological activities of ovine (o) and bovine (b) placental lactogens (PLs) and their mutated analogues were compared using several binding and in vitro bioassays. In almost all cases, the biological activities of these analogues mediated through rat (r) prolactin receptor (PRLR) showed little or no change, despite a remarkable decrease in their capacity to bind to the extracellular domain of rPRLR and despite compromised stability of the 2:1 complexes. These results indicate that mutations impairing the ability of oPL or bPL to form stable complexes with lactogenic receptors do not necessarily lead to a decrease in the biological activity, because the transient existence of the homodimeric complex is still sufficient to initiate the signal transduction. In contrast, oPL and bPL analogues completely, or almost completely, lost their ability to activate homologous PRLRs, and some of them even acted as site-2 antagonists. To explain the difference between the activity transduced through homologous and that transduced through heterologous PRLRs, we propose the novel term 'minimal time of homodimer persistence'.This concept assumes that in order to initiate the signal transduction, the associated kinase JAK2 has to be transphosphorylated and this requires a 'minimal time' of homodimer existence. In the case of homologous interaction between ruminant PLs and homologous PRLRs, this 'minimal time' is met, though the interaction with homologous PRLRs has a shorter half-life than that with heterologous PRLRs. Therefore oPL or bPL are active in cells possessing both homologous and heterologous PRLRs. Mutations of oPL or bPL lead to reduced affinity and, consequently, the 'time of homodimer persistence' is shortened. Although in the case of heterologous interaction the 'minimal time' is still sufficient to initiate the biological activity, in homologous interactions, which are already weaker than heterologous interactions, further destabilization of the complex shortens its persistence to below the 'minimal time', leading to full or partial loss of biological activity.

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Selective Modification of Recombinant Bovine Placental Lactogen by Site-directed Mutagenesis at Its C Terminus

Cited by 12 publications

References 49 publications

Novel Recombinant Analogues of Bovine Placental Lactogen

Novel Recombinant Analogues of Bovine Placental Lactogen

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

Mutations of ovine and bovine placental lactogens change, in different ways, the biological activity mediated through homologous and heterologous lactogenic receptors

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