Identification of gain-of-function variants of the human prolactin receptor

There is currently no known genetic disease linked to prolactin (PRL) or its receptor (PRLR) in humans. Recently, we identified three missense variants of the PRLR in patients presenting with breast tumors. Two of them (named PRLR(I146L) and PRLR(I76V)) had been reported earlier, but failed to draw much attention because the eventual impact of these substitutions on receptor properties remained unknown. In this chapter, we describe the various bioassays (cell types and readouts) that led to the discovery that both variants exhibit gain-of-function properties. Reconstituted cell models involving Ba/F3, HEK293, and MCF-7 cell lines all highlighted the constitutive, PRL-independent potency of PRLR(I146L) to trigger downstream signaling, leading to antiapoptotic and proliferation properties. The lower level of basal activity of PRLR(I76V) could be demonstrated only in the very sensitive Ba/F3 cell assay. While comparative analysis of ligands is a routine procedure in many labs, comparison of receptor variants de facto imposes the use of different cell clones (or population) in which each receptor variant is expressed individually. This is more delicate, as one must ensure that differences in biological responses really reflect differences in the intrinsic properties of receptor variants, and not any feature of cell clones/populations that are used, which could bias the interpretation.