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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

Endokrinologikum Hamburg, 20251 Hamburg, Germany

Address all correspondence and requests for reprints to: Birgit Gellersen, Ph.D., Endokrinologikum Hamburg, Falkenried 88, 20251 Hamburg, Germany. E-mail: gellersen{at}endokrinologikum.com.

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.

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