Restricted accessResearch articleFirst published online 2005-12
Immunofluorescent Localization of a Novel Progesterone Receptor(s) in a T47D-Y Breast Cancer Cell Line Lacking Genomic Progesterone Receptor Expression
Objective:To identify a novel nongenomic progesterone receptor (PR), PR-M, in T47D-Y breast cancer cells lacking genomic PR expression.
Methods:Immunofluorescent staining of T47D and T47D-Y breast cancer cells with selective anti-PR antibodies and ligand binding. Transient transfection of breast cancer cells with a cDNA expressing PR-M with a carboxy terminal green fluorescent protein.
Results:In the T47D-Y cell line, lacking expression of genomic PR, plasma membrane-bound and intracellular PR(s) are identified with anti-PR antibodies directed to the hormone-binding domain but not with a antibody direced to the amino terminus. A plasma membrane PR is also evident by immunofluorescent ligand binding. Expression of a novel truncated PR (PR-M) tagged with green fluorescent protein showed intracellular localization.
Conclusions:These studies support the expression of novel, truncated PR (PR-M) in a breast cancer cell line known to lack expression of genomic PR. This observation raises the possibility of progesterone action in breast cancer cells classically considered nonresponsive due to lack of genomic PR expression.
1. Conneely O, Mulac-Jercevic B, DeMayo F, Lydon J, O'Malley B. Reproductive functions of progesterone receptors. Recent Prog Horm Res2002;57:339-355.
2.
2. Misrahi M, Atger M, d'Auriol L, et al. Complete amino acid sequence of the human progesterone receptor deduced from cloned cDNA. Biochem Biophys Res Commun1987;143:740-748.
3.
3. Kastner P, Krust A, Turcotte B, et al. Two distinct estrogen-regulated promoters generate transcripts encoding the two functionally different progesterone receptor forms A and B. EMBO1990;9:1603-1614.
4.
4. Laraiskou A, Dupre A, Haccard O, Jessus C. From progesterone to active Cdc2 in Xenopus oocytes: A puzzling signaling pathway. Biol Cell2001;93:35-46.
5.
5. Tian J, Kim S, Heilig E, Ruderman J. Identification of XPR-1, a progesterone receptor required for Xenopus oocyte activation. Proc Natl Acad Sci U S A2000;97:14358-14363.
6.
6. Tesarik J, Mendoza C, Moos J, Carreras A. Selective expression of a progesterone receptor on the human sperm surface. Fertil Steril1992;58:784-792.
7.
7. Kirkman-Brown J, Punt E, Barrat C, Publicover S. Zona pellucida and progesterone-induced Ca2+ signaling and acrosome reaction in human spermatozoa. J Androl2002;23:306-315.
8.
8. Peluso JJ, Fernandez G, Pappalardo A, White BA. Characterization of a putative membrane receptor for progesterone in rat granulosa cells. Biol Reprod2001;65:94-101.
9.
9. Blackmore P, Im W, Bleasdale J. The cell surface progesterone receptor which stimulates calcium influx in human sperm is unlike the A-ring reduced steroid site on the GABAA receptor/chloride channel. Mol Cell Endocrinol1994;104:237-243.
10.
10. Peluso JJ, Fernandez G, Pappalardo A, White BA. Membraneinitiated events account for progesterone(apos)s ability to regulate intracellular free calcium levels and inhibit rat granulosa cell mitosis. Biol Reprod2002;67:379-385.
11.
11. Roldan E, Murase T, Shi Q. Exocytosis in spermatozoa in response to progesterone and zona pellucida. Science1994;266:1578-1581.
12.
12. Boonyaratanakornkit V, Scott M, Ribon V, et al. Progesterone receptor contains a proline-rich motif that directly interacts with SH3 domains and activates c-Src family tyrosine kinases. Mol Cell2001;8:269-280.
13.
13. Zhu Y, Rice C, Pang Y, Thomas P. Cloning, expression, and characterization of a membrane progestin receptor and evidence it is an intermediary in meiotic maturation of fish occytes. Proc Natl Acad Sci U S A2003;100:2231-2236.
14.
14. Zhu Y, Bond J, Thomas P. Identification, classification, and partial characterization of genes in humans and other vertebrates homologous to a fish membrane progestin receptor. Proc Natl Acad Sci U S A2003;100:2237-2242.
15.
15. Meyer C, Schmid R, Scriba PC, Wehling M. Purification and partial sequencing of high affinity progesterone-binding site(s) from porcine liver membranes. Eur Biochem1996;239:726-731.
16.
16. Gerdes D, Wehling M, Leube B, Falkenstein E. Cloning and tissue expression of two putative steroid membrane receptors. Biol Chem1998;379:907-911.
17.
17. Falkenstein E, Heck M, Gerdes D, et al. Specific progesterone binding to a membrane protein and related nongenomic effects on Ca2+-fluxes in sperm. Endocrinology1999;140:5999-6002.
18.
18. Saner K, Welter B, Zhang F, et al. Cloning and expression of a novel, truncated progesterone receptor. Mol Cell Endocrinol2003;200:155-163.
19.
19. Guiochon-Mantel A, Loosfelt H, Lescop P, et al. Mechanisms of nuclear localization of the progesterone receptor: Evidence for interaction between monomers. Cell1989;57:1147-1154.
20.
20. Sambrook J, Fritsch E, Maniatis T, eds. Strategies for cloning in plasmid vectors. In: Molecular Cloning. Vol. 1. Plainview, NY: Cold Spring Harbor Laboratory Press, 1989:1.53-1.73.
21.
21. Nielsen H, Engelbrecht J, Brunak S, Von Heijne G. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Engineer1997;10:1-6.
22.
22. Venter JC, Adams MD, Miers EW, Li PW, Mural RJ, Sutton GG. The sequene of the human genome. Science2001;291:1305-1351.
23.
23. Sartorius C, Shen T, Horwitz KB. Progesterone receptors A and B differentially affect the grwoth of estrogen-dependent human breast tumor xenografts. Breast Cancer Res Treat2003;79:287-299.
24.
24. Weiler PJ, Wiebe JP. Plasma membrane receptors of the cancer-regulating progesterone metabolities, 5α-pregnane-3,20-dione and 3α-hydroxy-4-pregnen-20-one in MCF-7 breast cancer cells. Biochem Biophys Res Commun2000;272:731-737.
25.
25. Wan Y, Coxe KK, Thackray VG, Housely PR, Nordeen SK. Separable features of the ligand-binding domain determine the differential subcellular localization and ligand-binding specificity of glucocorticoid receptor and progesterone receptor. Mol Endocrinol2001;15:17-31.
26.
26. Htun H, Holth LT, Walker D, Davie JR, Hager GL. Direct visualization of the human estrogen receptor alpha reveals a role for ligand in the nuclear distribution of the receptor. Mol Biol Cell1999;10:471-486.
27.
27. Faivre E, Skildum A, Pierson-Mullany P, Lange CA. Integration of progesterone receptor mediated rapid signaling and nuclear actions in breast cancer cell models: Role of mitogen-activated protein kinases and cell cycle regulators. Steroids2005;70:418-426.
28.
28. Migliaccio A, Piccolo D, Castoria G, et al. Activation of the Src/p21ras/Erk pathway by progesterone receptor via cross-talk with estrogen receptor. EMBO J. 1998;17:2008-2018.
29.
29. Hodges YK, Richer JK, Hortwitz KB, Hortwitz LD. Variant estrogen and progesterone receptor messages in human vascular smooth muscle. Circulation1999;99:2688-2693.
30.
30. Richer J, Lange C, Wierman A, et al. Progesterone receptor variants found in breast cells repress transcription by wild-type receptor. Breast Cancer Res Treat1998;48:231-241.
31.
31. Tetel M, Jung S, Carbajo P, Ladtkow T, Skafar D, Edwards D. Hinge and amino-terminal sequences contribute to solution dimerization of human progesterone receptor. Mol Endocrinol1997;11:1114-1128.
32.
32. Kirkman-Brown J, Bray C, Stewart P, Barratt C, Publicover S. Biphasic elevation of [Ca2+]i in individual human spermatozoa exposed to progesterone. Dev Biol2000;222:326-335.
33.
33. Arnoult C, Villaz M, Florman H. Pharmacological properties of the T-type Ca2+ current of mouse spermatogenic cells. Mol Pharmacol1998;53:1104-1111.
34.
34. Thomas P, Meizel S. Phosphatidylinositol-4,5-bisphosphate hydrolysis in human sperm stimulated with follicular fluid or progesterone is dependent on Ca2+ influx. Biochem J1989;264:539-546.
35.
35. Kobori H, Miyazaki S, Kuwabara Y. Characterization of intracellular Ca2+ increase in response to progesterone and cyclic nucleotides in mouse spermatozoa. Biol Reprod2000;63:113-120.
36.
36. Barbagallo M, Dominguez L, Licata G, et al. Vascular effects of progesterone: Role of cellular regulation. Hypertension2001;37:142-147.
37.
37. Bielefeldt K, Waite L, Abboud F, Conklin J. Nongenomic effects of progesterone on human intestinal smooth muscle cells. Am J Physiol1996;271:G370-G376.
38.
38. Berridge M, Bootman M, Roderick H. Calcium signalling: Dynamics, homeostasis and remodelling. Nature Rev2003;4:517-529.
39.
39. Kahl CR, Means AR. Regulation of cell cycle progression by calcium/calmodulin-dependent pathways. Endocr Rev2003;24:719-736.
