Systematic expression analysis and antibody screening do not support the existence of naturally occurring progesterone receptor (PR)-C, PR-M, or other truncated PR isoforms.
Functional progesterone withdrawal associated with human parturition has been ascribed to various mechanisms modulating the function of the classical progesterone receptors (PRs), B and A, in utero. These include up-regulation of the inhibitory PR-C isoform, described as a 60-kDa protein occurring from translation initiation at codon 595. Our initial attempts to detect PR-C yielded uninterpretable results. To systematically validate antibodies for immunodetection of PR isoforms, we generated expression vectors for PR variants originating from putative start codons AUG-289, -301, -595, -632, and -692 in addition to those for PR-B and PR-A, and for alternative splice variants PR-T, PR-S, and PR-M. All constructs were subjected to in vitro and in vivo translation and immunoblotting with a panel of 13 PR antibodies. Antibodies raised against full-length PR were generally not capable of detecting N-terminally truncated forms, whereas C-terminal antibodies did not or only weakly reacted with PR-B and PR-A but produced prominent nonspecific signals. Thus, immunodetection of N-terminally truncated PR isoforms is prone to artifacts. Proteins of about 64 kDa were expressed from PR-289 and -301, but no corresponding endogenous forms were observed. PR-T, PR-S, and PR-M cDNAs yielded no detectable translation products. No protein was translated from AUG-595 in our PR-C expression vector unless a Kozak sequence was introduced, and the product was not 60 but 38 kDa in size. Thus, the 60-kDa protein called PR-C does not originate from AUG-595 and is not a naturally occurring PR isoform.
Samalecos
Annemarie
A
Endokrinologikum Hamburg, Falkenried 88, 20251 Hamburg, Germany.
Gellersen
Birgit
B
D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D005260:Female; D020869:Gene Expression Profiling; D006801:Humans; D007118:Immunoassay; D009215:Myometrium / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D022821:RNA Splice Sites / Q000235:genetics / Q000502:physiology; D011980:Receptors, Progesterone / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D024363:Transcription Initiation Site / Q000502:physiology
D000906:Antibodies; D020033:Protein Isoforms; D022821:RNA Splice Sites; D011980:Receptors, Progesterone