Mechanism for Ordered Receptor Binding by Human Prolactin

Sivaprasad, Umasundari; Canfield, Jeffrey M.; Brooks, Charles L.

doi:10.1021/bi049333p

Cited by 30 publications

(44 citation statements)

References 30 publications

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“…Based on fluorescence resonance energy transfer data, an allosteric coupling of BS1 and BS2, involving receptor-induced conformational changes in PRL, was suggested (34). However, no detailed molecular mechanism was formulated, and the extent to which PRL actually undergoes significant structural rearrangements when interacting with the receptor chain via BS1 has been a matter of some debate.…”

Section: Discussionmentioning

confidence: 99%

Crystal Structure of a Prolactin Receptor Antagonist Bound to the Extracellular Domain of the Prolactin Receptor

Svensson

Bondensgaard

Nørskov-Lauritsen

et al. 2008

Journal of Biological Chemistry

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The crystal structure of the complex between an N-terminally truncated G129R human prolactin (PRL) variant and the extracellular domain of the human prolactin receptor (PRLR) was determined at 2.5 Å resolution by x-ray crystallography. This structure represents the first experimental structure reported for a PRL variant bound to its cognate receptor. The binding of PRL variants to the PRLR extracellular domain was furthermore characterized by the solution state techniques, hydrogen exchange mass spectrometry, and NMR spectroscopy. Compared with the binding interface derived from mutagenesis studies, the structural data imply that the definition of PRL binding site 1 should be extended to include residues situated in the N-terminal part of loop 1 and in the C terminus. Prolactin (PRL)4 is a protein hormone secreted by the anterior pituitary in vertebrates and possesses physiological functions of remarkable diversity, including effects on reproduction, lactation, and growth. PRL belongs to a family of homologous proteins comprising PRL, growth hormone (GH), and placental lactogen (PL). The biological effects associated with this cytokine family are mediated by two distinct classes of cell surface receptors, the PRL receptors (PRLR) and the GH receptors (GHR). The PRL/PL/GH biology is governed by a delicate balance between receptor cross-reactivity and selectivity; PRL and PL bind selectively to PRLR, whereas GH is capable of binding both PRLR and GHR.After being proposed more than a decade ago (1), hormoneinduced receptor dimerization became generally accepted as the model for cytokine receptor activation. For the PRL family members, the model describes the signaling molecular entity as a ternary complex between one hormone molecule and a receptor homodimer assembled in a strictly sequential and hormonedependent fashion; first the hormone ligand engages via binding site 1 (BS1) in high affinity binding to one receptor chain forming a 1:1 hormone-receptor complex. This complex constitutes the template for binding a second, identical receptor molecule via binding site 2 (BS2), resulting in the active 1:2 complex. However, the model has been challenged by an increasing body of experimental evidence, initially reported for the homologous human erythropoietin receptor (2) and later for GHR (3) and PRLR (4). These studies suggest that preformed, inactive dimers exist in the absence of hormone. Thus, receptor dimerization is a necessary but not sufficient event for receptor activation and, notably, not strictly ligand-dependent. For both human erythropoietin receptor (5) and GHR (3), mechanistic models have been proposed, where receptor activation involves relative rotations and movements of receptor subunits induced by hormone binding. Allosteric reorganization of the intracellular receptor domains brings associated JAK2 kinases into close proximity, allowing their activation by cross-phosphorylation. This initial activation step triggers a cascade of molecular events leading to the functional receptor response (6).The...

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Section: Discussionmentioning

confidence: 99%

Crystal Structure of a Prolactin Receptor Antagonist Bound to the Extracellular Domain of the Prolactin Receptor

Svensson

Bondensgaard

Nørskov-Lauritsen

et al. 2008

Journal of Biological Chemistry

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“…19 More likely, as recently suggested, the affinity for the second receptor molecule increases significantly when site 1 is occupied. 2 Still, site 1 has a higher affinity for hPRLR-ECD compared to the induced affinity of site 2, as native PAGE or gel filtration at sub-stoichiometric amounts of hPRLR-ECD and hPRL observes no ternary complex. At more than one equivalent of hPRLR-ECD, both binary and ternary complexes are observed by native-PAGE and gel filtration.…”

Section: Receptor Binding Studiesmentioning

confidence: 97%

“…Upon binding of a receptor molecule at site 1, the affinity for binding of a second receptor molecule is substantially increased approaching the affinity of site 1. 2,19 From mutagenesis studies, Ala22, Arg21, Tyr28, Arg125 and Gly129 were shown to be important for receptor binding. 9,29,36,38 Inspection of the putative binding site from an early structural model of PRL suggested a set of small residues to be important for maintaining the geometry of the site.…”

Section: Binding Sitementioning

confidence: 99%

“…Based on fluorescence resonance energy transfer studies binding of the first receptor subunit to hPRL has been hypothesized to induce a conformational change in the hormone that increases the affinity for binding of the second receptor subunit. 2 Site 2 is thus considered not competent for receptor binding in the free hormone. 2 PRL is a member of the long-chain cytokine family and is closely related to growth hormone (GH) and placental lactogen (PL), with both of which it shares 23% sequence identity.…”

Section: Introductionmentioning

confidence: 99%

“…2 Site 2 is thus considered not competent for receptor binding in the free hormone. 2 PRL is a member of the long-chain cytokine family and is closely related to growth hormone (GH) and placental lactogen (PL), with both of which it shares 23% sequence identity. Whereas GH is able to bind to the GH receptor (GHR) and for primates GH to PRLR, PRL (and non-ruminant PL) only binds PRLR.…”

Section: Introductionmentioning

confidence: 99%

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Solution Structure of Human Prolactin

Teilum

Hoch

Goffin

et al. 2005

Journal of Molecular Biology

114

110

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We report the solution structure of human prolactin determined by NMR spectroscopy. Our result is a significant improvement over a previous structure in terms of number and distribution of distance restraints, regularity of secondary structure, and potential energy. More significantly, the structure is sufficiently different that it leads to different conclusions regarding the mechanism of receptor activation and initiation of signal transduction. Here, we compare the structure of unbound prolactin to structures of both the homologue ovine placental lactogen and growth hormone. The structures of unbound and receptor bound prolactin/ placental lactogen are similar and no noteworthy structural changes occur upon receptor binding. The observation of enhanced binding at the second receptor site when the first site is occupied has been widely interpreted to indicate conformational change induced by binding the first receptor. However, our results indicate that this enhanced binding at the second site could be due to receptor-receptor interactions or some other free energy sources rather than conformational change in the hormone. Titration of human prolactin with the extracellular domain of the human prolactin receptor was followed by NMR, gel filtration and electrophoresis. Both binary and ternary hormone-receptor complexes are clearly detectable by gel filtration and electrophoresis. The binary complex is not observable by NMR, possibly due to a dynamic equilibrium in intermediate exchange within the complex. The ternary complex of one hormone molecule bound to two receptor molecules is on the contrary readily detectable by NMR. This is in stark contrast to the widely held view that the ternary prolactinreceptor complex is only transiently formed. Thus, our results lead to improved understanding of the prolactin-prolactin receptor interaction.

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Development of prolactin receptor antagonists with reduced pH‐dependence of receptor binding

Hansen¹,

Olsen²,

Bernichtein³

et al. 2010

J of Molecular Recognition

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The cytokine hormone prolactin has a vast number of diverse functions. Unfortunately, it also exhibits tumor growth promoting properties, which makes the development of prolactin receptor antagonists a priority. Prolactin binds to its cognate receptor with much lower affinity at low pH than at physiological pH and since the extracellular environment around solid tumors often is acidic, it is desirable to develop antagonists that have improved binding affinity at low pH. The pK(a) value of a histidine side chain is ∼6.8 making histidine residues obvious candidates for examination. From evaluation of known molecular structures of human prolactin, of the prolactin receptor and of different complexes of the two, three histidine residues in the hormone-receptor binding site 1 were selected for mutational studies. We analyzed 10 variants by circular dichroism spectroscopy, affinity and thermodynamic characterization of receptor binding by isothermal titration calorimetry combined with in vitro bioactivity in living cells. Histidine residue 27 was recognized as a central hot spot for pH sensitivity and conservative substitutions at this site resulted in strong receptor binding at low pH. Pure antagonists were developed earlier and the histidine mutations were introduced within such background. The antagonistic properties were maintained and the high affinity at low pH conserved. The implications of these findings may open new areas of research in the field of prolactin cancer biology.

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Mechanism for Ordered Receptor Binding by Human Prolactin

Cited by 30 publications

References 30 publications

Crystal Structure of a Prolactin Receptor Antagonist Bound to the Extracellular Domain of the Prolactin Receptor

Crystal Structure of a Prolactin Receptor Antagonist Bound to the Extracellular Domain of the Prolactin Receptor

Solution Structure of Human Prolactin

Development of prolactin receptor antagonists with reduced pH‐dependence of receptor binding

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