Unmodified prolactin (PRL) and S179D PRL-initiated bioluminescence resonance energy transfer between homo- and hetero-pairs of long and short human PRL receptors in living human cells.
We have used bioluminescence resonance energy transfer (BRET) to examine the interaction between human prolactins (PRLs) and the long (LF) and two short isoforms (SF1a and SF1b) of the human PRL receptor in living cells. cDNA sequences encoding the LF, SF1a, and SF1b were subcloned into codon-humanized vectors containing cDNAs for either Renilla reniformis luciferase (Rluc) or a green fluorescent protein (GFP(2)) with a 12- or 13-amino acid linker connecting the parts of the fusion proteins. Transfection into human embryonic kidney 293 cells demonstrated maintained function of Rluc and GFP(2) when linked to the receptors, and confocal microscopy demonstrated the localization of tagged receptors in the plasma membrane by 48 h after transfection. All three tagged receptors transduced a signal, with the LF and SF1a stimulating, and SF1b inhibiting, promoter activity of an approximately 2.4-kb beta-casein-luc construct. Both unmodified PRL (U-PRL) and the molecular mimic of phosphorylated PRL, S179D PRL, induced BRET with all combinations of long and short receptor isoforms except SF1a plus SF1b. No BRET was observed with the site two-inactive mutant, G129R PRL. This is the first demonstration, 1) that species homologous PRL promotes both homo- and hetero-interaction of most long and short PRLR pairs in living cells, 2) that both U-PRL and S179D PRL are active in this regard, and 3) that there is some aspect of SF1a-SF1b structure that prevents this particular hetero-receptor pairing. In addition, we conclude that preferential pairing of different receptor isoforms is not the explanation for the different signaling initiated by U-PRL and S179D PRL.
Tan
Dunyong
D
Division of Biomedical Sciences, University of California-Riverside, Riverside, CA 92521-0121, USA.
Johnson
David A
DA
Wu
Wei
W
Zeng
Lingfang
L
Chen
Yen Hao
YH
Chen
Wen Y
WY
Vonderhaar
Barbara K
BK
Walker
Ameae M
AM
D019943:Amino Acid Substitution; D002364:Caseins / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D006801:Humans; D008163:Luminescent Measurements; D011388:Prolactin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D011981:Receptors, Prolactin / Q000737:chemistry / Q000378:metabolism*
D002364:Caseins; D020033:Protein Isoforms; D011981:Receptors, Prolactin; D011388:Prolactin