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Published on: January 28, 2013
Investigation of prolactin receptor activation and blockade using time-resolved fluorescence resonance energy
Estelle Tallet1, Isabelle Fernandez, Chi Zhang
1INSERM, U845, Centre de Recherche "Croissance et Signalisation", Equipe "Physiopathologie des hormones PRL/GH" Paris, France.
Abstract:
The prolactin receptor (PRLR) is emerging as a therapeutic target in oncology. Knowledge-based drug design led to the development of a pure PRLR antagonist (Del1-9-G129R-hPRL) that was recently shown to prevent PRL-induced mouse prostate tumorogenesis. In humans, the first gain-of-function mutation of the PRLR (PRLR(I146L)) was recently identified in breast tumor patients. At the molecular level, the actual mechanism of action of these two novel players in the PRL system remains elusive. In this study, we addressed whether constitutive PRLR activation (PRLR(I146L)) or PRLR blockade (antagonist) involved alteration of receptor oligomerization and/or of inter-chain distances compared to unstimulated and PRL-stimulated PRLR. Using a combination of various biochemical and spectroscopic approaches (co-IP, blue native electrophoresis, BRET(1)), we demonstrated that preformed PRLR homodimers are altered neither by PRL- or I146L-induced receptor triggering, nor by antagonist-mediated blockade. These findings were confirmed using a novel time-resolved fluorescence resonance energy transfer (TR-FRET) technology that allows monitoring distance changes between cell surface tagged receptors. This technology revealed that PRLR blockade or activation did not involve detectable distance changes between extracellular domains of receptor chains within the dimer. This study merges with our previous structural investigations suggesting that the mechanism of PRLR activation solely involves intermolecular contact adaptations leading to subtle intramolecular rearrangements.
Insights
Prolactin receptor (PRLR) antagonists and mutations do not alter receptor dimerization or distances. This suggests PRLR activation involves subtle intramolecular changes, not altered receptor spacing.
Area of Science:
- Molecular and Cellular Biology
- Biochemistry
- Oncology
Background:
- The prolactin receptor (PRLR) is a key target in cancer therapy.
- A novel PRLR antagonist (Del1-9-G129R-hPRL) inhibits PRL-induced tumors.
- A gain-of-function PRLR mutation (PRLR(I146L)) is linked to human breast tumors.
Purpose of the Study:
- To investigate the molecular mechanism of PRLR activation and blockade.
- To determine if PRLR activation (PRLR(I146L)) or blockade (antagonist) affects receptor oligomerization or inter-chain distances.
Main Methods:
- Biochemical and spectroscopic techniques including co-immunoprecipitation (co-IP), blue native electrophoresis, and bioluminescence resonance energy transfer (BRET).
- Novel time-resolved fluorescence resonance energy transfer (TR-FRET) to monitor distance changes between cell surface tagged receptors.
Main Results:
- Preformed PRLR homodimers are not altered by PRL stimulation, I146L mutation, or antagonist blockade.
- TR-FRET analysis confirmed no detectable changes in distances between extracellular domains of receptor chains within the dimer.
- PRLR activation or blockade does not involve alterations in receptor oligomerization or significant inter-chain distance changes.
Conclusions:
- PRLR activation and blockade mechanisms do not rely on changes in receptor dimerization or inter-chain distances.
- Findings suggest PRLR activation involves subtle intramolecular rearrangements rather than altered receptor spacing.
- This study provides crucial insights into the molecular basis of PRLR function in normal and pathological conditions.
