Cloning and characterization of an ovine intracellular seven transmembrane receptor for progesterone that mediates calcium mobilization.
Classically, progesterone has been thought to act only through the well-known genomic pathway involving hormone binding to nuclear receptors (nPR) and subsequent modulation of gene expression. However, there is increasing evidence for rapid, nongenomic effects of progesterone in a variety of tissues in mammals, and it seems likely that a membrane PR (mPR) is causing these events. The objective of this study was to isolate and characterize an ovine mPR distinct from the nPR. A cDNA clone was isolated from ovine genomic DNA by PCR. The ovine mPR is a 350-amino acid protein that, based on computer hydrophobicity analysis, possesses seven transmembrane domains and is distinct from the nPR. Message for the ovine mPR was detected in hypothalamus, pituitary, uterus, ovary, and corpus luteum by RT-PCR. In CHO cells that overexpressed a mPR-green fluorescent protein fusion protein, the ovine mPR was localized to the endoplasmic reticulum and not the plasma membrane. Specific binding of 3H-progesterone to membrane fractions was demonstrated in CHO cells that expressed the ovine mPR but not in nontransfected cells. Furthermore, progesterone and 17 alpha-hydroxy-progesterone stimulated intracellular Ca2+ mobilization in CHO cells that expressed ovine mPR in Ca2+-free medium (P < 0.05) but not in CHO cells transfected with empty vector. This rise in intracellular Ca2+ is believed to be from the endoplasmic reticulum as intracellular Ca2+ mobilization is absent when mPR transfected cells are first treated with thapsigargin to deplete Ca2+ stores from the endoplasmic reticulum. Isolation, identification, tissue distribution, cellular localization, steroid binding, and a functional response for a unique intracellular mPR in the sheep are presented.
Ashley
R L
RL
Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523, USA.
Clay
C M
CM
Farmerie
T A
TA
Niswender
G D
GD
Nett
T M
TM
D019326:17-alpha-Hydroxyprogesterone / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D003001:Cloning, Molecular; D003338:Corpus Luteum / Q000737:chemistry; D006224:Cricetinae; D003412:Cricetulus; D018076:DNA, Complementary / Q000302:isolation & purification; D004721:Endoplasmic Reticulum / Q000737:chemistry; D005260:Female; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics; D007031:Hypothalamus / Q000737:chemistry; D008969:Molecular Sequence Data; D010053:Ovary / Q000737:chemistry; D010902:Pituitary Gland / Q000737:chemistry; D011374:Progesterone / Q000378:metabolism / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D011980:Receptors, Progesterone / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D011993:Recombinant Fusion Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012756:Sheep; D019284:Thapsigargin / Q000494:pharmacology; D014162:Transfection; D014316:Tritium; D014599:Uterus / Q000737:chemistry
D018076:DNA, Complementary; D012333:RNA, Messenger; D011980:Receptors, Progesterone; D011993:Recombinant Fusion Proteins; D014316:Tritium; D049452:Green Fluorescent Proteins; D011374:Progesterone; D019284:Thapsigargin; D019326:17-alpha-Hydroxyprogesterone; D002118:Calcium