FP39-4 Identifying Residues of the Prolactin Receptor Extracellular Domain that Control Intracellular Signaling

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: FP39-Pituitary
Monday, June 17, 2013: 10:45 AM-11:15 AM
Presentation Start Time: 11:00 AM
Room 133 (Moscone Center)

Poster Board MON-117
Chi Zhang*1, Mads Nygaard2, Gitte W. Haxholm2, Isabelle Broutin3, Birthe B. Kragelund2 and Vincent Goffin1
1Inserm U845, Paris, France, 2University of Copenhagen, 3Faculté de Pharmacie, Université Paris Descartes
Prolactin receptor (PRLR) signaling is triggered by the formation of a heterotrimeric complex involving one ligand and two receptor moieties (1). However, the mechanisms that propagate the signal from the extracellular domain (ECD) to the intracellular domain (ICD) of the receptor remain poorly understood. Ligand-independent PRLR signaling can be achieved by deleting the ECD-D2 domain, suggesting that the latter intrinsically blocks structural rearrangements of the ICD that are required to trigger signaling (2). Furthermore, we recently identified a single amino acid substitution (I146L) within the D2 domain that also conferred ligand-independent activity (3). An NMR study of D2 domain of WT-PRLR showed that in the unbound state, both Trp of the conserved WS motif adopted a T-stack conformation that stabilized the receptor off-state while in the ligand-bound state these form a Trp-Arg ladder characteristic of the receptor on-state (4). Interestingly, chemical shift perturbation studies pointed to structural rearrangements involving Ile146 and the receptor-receptor dimerization interface (site 3), suggesting that these two features may participate in PRLR activation. To address this hypothesis, we generated two groups of mutants: the first one involved substitution of various residues for Ile146, and the second one involved replacements of several site 3 residues identified based on the crystal structure of the heterotrimeric PRL/PRLR complex (1). Cell-based assays showed that manipulation of Ile146 profoundly altered the intrinsic PRLR properties in a mutation-specific manner, including PRL-responsiveness, ligand-independent activity and signaling cascade specificity. Preliminary structural analyses of cognate ECD/D2 domains are suggestive of novel T-stack conformation disturbances in some mutants, in this case involving aromatic residues of site 3, suggesting a role for Ile146 in maintaining D2 folding as well as binding competency. Although none of the mutations involving site 3 conferred ligand-independent activity, they profoundly altered PRLR properties regarding species-specificity for ligand binding and signaling cascade selectivity. Their analysis by NMR is ongoing. Taken together, these data point to an important role of Ile146 and of site 3 residues in the regulation of, and the selectivity in PRLR signaling, which may involve the control of structural rearrangements that are necessary to propagate the signal from the ECD to the ICD of the PRLR.

(1) Broutin et al, (2010), JBC, 285(11):8422-33. (2) Tan et al, (2008), Biochemistry, 47(1): 479–489. (3) Bogoral et al, (2008), Proc Natl Acad Sci U S A, 105(38):14533-8.(4) Dagil et al, (2012), Structure, 20(2):270-82.

Nothing to Disclose: CZ, MN, GWH, IB, BBK, VG

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm

Sources of Research Support: CZ supported by a fellowship from Fondation ARC pour la recherche sur le cancer (DOC20110603059 ).