S2 deletion variants of human PRL receptors demonstrate that extracellular domain conformation can alter conformation of the intracellular signaling domain.
Using spacers between the C-termini of the long (LF) or short (SF) human prolactin receptors and luciferase/GFP such that bioluminescence resonance energy transfer (BRET) occurred minimally in intact versions of these receptors in the absence of ligand, we have monitored the BRET signal after deletion of regions of the extracellular domain (ECD). Deletion of S2 produced ligand-independent BRET for only those pairings normally occurring in the presence of ligand with the intact receptor. Deletion of the similarly sized S1, or S1 plus S2, produced no ligand-independent or -dependent BRET. When deleted receptors were transfected into human breast (T47D) or prostate (DU145) cancer cells incubated in the absence of added prolactin (PRL) and presence of anti-PRL, expression of the DeltaS2LF resulted in increased cell number, whereas expression of the intact receptor did not. When endogenous beta-casein expression was examined in T47D cells, the DeltaS2LF and DeltaS2F1a both showed ligand-independent activation of transcription, again not duplicated by the intact receptor. Paired with evidence in the literature for predimerization of PRLRs, these results demonstrate that altered ECD conformation, and not just a change in bulk, produces altered conformation of the intracellular signaling region of the receptors, supporting the concept that ligand binding to the ECD of intact predimerized receptors could initiate signaling. In addition, the current work supports a dual proliferative and differentiative role for the LF receptor, but only a differentiative role for the SF1a receptor. Naturally occurring DeltaS2 PRL receptors (PRLR) were also found in normal and cancerous human cells. This additionally suggests a heretofore unappreciated ligand-independent role for PRLRs.
Tan
Dunyong
D
Division of Biomedical Sciences, University of California, Riverside, California 92521, USA.
Huang
Kuang Tzu
KT
Ueda
Eric
E
Walker
Ameae M
AM
D015153:Blotting, Western; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004735:Energy Transfer; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods; D018613:Microscopy, Confocal; D011388:Prolactin / Q000494:pharmacology; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011981:Receptors, Prolactin / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017384:Sequence Deletion; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*; D014162:Transfection
D011981:Receptors, Prolactin; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D011388:Prolactin; D008156:Luciferases