Objective:Regulatory guanine nucleotide-binding proteins (G proteins) play key roles in the stimulus-response coupling of many important biological systems. Recent studies from our laboratory suggest a functional role for many G proteins in the human placenta. However, the expression of these proteins has not yet been reported. Therefore, the aims of this investigation were to identify the expression of placental G protein alpha subunits in human placenta, and to study their level of variation during pregnancy.
Methods:Western and Northern blot analyses were performed on membrane protein and mRNA preparations, respectively, of human placentas from the first (7-11 weeks), second (16-19 weeks), and third (term) trimesters.
Results:The autoradiographs of both proteins and mRNA showed differential expression of placental G proteins during pregnancy. Thus, the relative levels of Gαi2 and Gαi3 subunits were highest during the first trimester, whereas to differences were observed between second-trimester and term placentas for both subunits. The levels of placental Gαo and Gαs subunits stayed relatively stable during pregnancy.
Conclusion:The demonstration of human placental Gα protein expression during pregnancy provides new insight into the components of the signal transduction machinery within trophoblast. However, the physiologic significance of the variations of placental Gαi protein expression during pregnancy remains to be investigated.
1. Evans T, Brown ML, Fraster ED, Northup JK. Purification of the major GTP-binding proteins from human placental membranes. J Biol Chem1986;261:7052-7059.
2.
2. Tamir A, Fawzi AB, Northup JK. Unique guanine nucleotide-binding properties of the human placental GTP-binding protein Gp. Biochemistry1990;29:6947-6954.
3.
3. Petit A, Gallo-Payer N, Vaillancourt C, Bellabarba D, Lehoux J-G, Bélisle S. A role for extracellular calcium in the regulation of placental lactogen release by angiotensin-II and dopamine in human term trophoblastic cells. J Clin Endocrinol Metab1993;77:670-676.
4.
4. Petit A, Guillon G, Pantaloni C, et al. An islet-activating protein-sensitive G-protein is involved in dopamine inhibition of both angiotensin-stimulated inositol phosphate production and human placental lactogen release in human trophoblastic cells. J Clin Endocrinol Metab1990;71:1573-1580.
5.
5. Petit A, Gallo-Payet N, Bellabarba D, Lehoux L-G, Bélisle S. The modulation of placental lactogen release by opioids: a role for extracellular calcium. Mol Cell Endocrinol1993;90:165-170.
6.
6. Tencé M, Petit A. Characterization of angiotensin II binding sites in the human term placenta. Mol Cell Endocrinol1989;63:111-119.
7.
7. Porthé G, Francés B, Verrier B, Cros J, Meunier JC. The kappa-opioid receptor from human placenta: Hydrodynamic characteristics and evidence for its association with a G protein. Life Sci1988;43:559-567.
8.
8. ElMabrouk M, Bouvier C, Simoneau L, Savard R, Lafond J. Asymetrical distribution of G proteins in syncytiotrophoblastic brush border and basal plasma membranes in human placenta. Abstracts of the 75th Annual Meeting of the Endoctrine Society, Las Vegas, 1993:115-115.
9.
9. Tan L, Petit A. Inactivation of human placental aromatase by 6α and 6β-hydroperoxyandrostenedione. Biochem Biophys Res Commun1985;128:613-620.
10.
10. Painson J-C, Wenger T, Lagacé G, Masson ND, Collu R. Cellular distribution of G protein Goα in pituitary lactotrophs: Effects of dopamine. J Neuroendocrinol1994;6:447-455.
11.
11. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem1951;193:265-275.
12.
12. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature1970;227:680-685.
13.
13. Maniatis T, Fritsch EF, Sambrook J. Transformation of Escherichia coli by plasmid DNA. In: Molecular cloning. New York: Cold Spring Harbor Laboratory. 1982;249-251.
14.
14. Jones DT, Reed RR. Molecular cloning of five GTP-binding protein cDNA species from rat olfactory neuroepithelium. J Biol Chem1987;262:14241-14249.
15.
15. Promega protocols and applications guide. Second edition. Madison, Wisconsin: Promega Corporation, 1991:106-106.
16.
16. Chomcynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem1987;162:156-159.
17.
17. Maniatis T, Fritsch EF, Sambrook J. Extraction, purification and analysis of mRNA from eucaryotic cells. In: Molecular cloning. New York: Cold Spring Harbor Laboratory, 1982:203-203.
18.
18. Hepler JR, Gilman AG. G proteins. Trends in Biochemical Sci1992;17:383-387.
19.
19. Kenton P, Jackson PM. GTP-Binding proteins associated with the human placental syncytiotrophoblast plasma membrane. Placenta1994;15:1-11.
20.
20. Figgs LW. Guanosine nucleotide binding in human placental syncytiotrophoblast membranes and comparative regulation of adenylate cyclase in syncytiotrophoblast, turkey erythrocyte, and bovine calf testes membranes by guanosine-5′-triphosphate. Comp Biochem Physiol1988;89B:119-125.
21.
21. Matsubara S, Tamada T, Saito T. Ultracytochemical localizations of adenylate cyclase, guanylate cyclase, and cyclic 3′,5′-nucleotide phosphodiesterase activity on the trophoblast in the human placenta. Direct histochemical evidence. Histochemistry1987;87:505-509.
22.
22. Harman I, Costello A, Sane A, Handwerger S. Cyclic adenosine-3′:5′-monophosphate stimulates the acute release of placental lactogen from human trophoblast cells. Endocrinology1987;121:59-63.
23.
23. Petit A, Gallo-Payet N, Lehoux J-G, Bellabarba D, Bélisle S. Adenosine 3′:5′-cyclic monophosphate (cAMP) is not the mediator of kappa opiate effect on human placental lactogen release. Life Sci1991;49:465-472.
24.
24. Cattini PA, Klassen M, Nachtigal M. Regulation of human chorionic somatomammotropin gene expression in rat pituitary tumor cells. Mol Cell Endocrinol1988;60:217-224.
25.
25. Hussa RO, Patillo RA, Ruckert ACF, Scheuerman KW. Effects of butyrate and dibutyryl cyclic AMP on hCG-secreting trophoblastic and non-trophoblastic cells. J Clin Endocrinol Metab1978;46:69-76.
26.
26. Nulsen JC, Woolkales MJ, Kopf GS, Strauss JF III. Adenylate cyclase in human cytotrophoblasts: Characterization and its role in modulating human chorionic gonadotropin secretion. J Clin Endocrinol Metab1988;66:258-265.
27.
27. Ringler GE, Kao L-C, Miller WL, Strauss JF III. Effects of 8-bromo-cAMP on expression of endocrine functions by cultured human trophoblast cells. Regulation of specific mRNA. Mol Cell Endocrinol1989;61:13-21.
29. Yatani A, Imoto Y, Codina J, Hamilton SL, Brown AM, Birnbaumer L. The stimulatory G protein of adenylate cyclase, Gs, also stimulates dihydropyridine-sensitive Ca2+ channels: Evidence for direct regulation independent of phosphorylataion. J Biol Chem1988;263:9887-9895.
30.
30. Imoto Y, Yatani A, Reeves JP, Codina J, Birnbaumer L, Brown AM. The α subunit of Gs directly activates cardiac calcium channels in lipid bilayers. Am J Physiol1988;255:H722-H728.
31.
31. Mattera R, Graziano MP, Yatani A, et al. Splice variants of the α subunit of the G protein Gs activate both adenylyl cyclase and calcium channels. Science1989;243:804-807.
32.
32. Meuris S, Polliotti B, Robyn C, Lebrun P. Ca2+ entry through L-type voltage-sensitive Ca2+ channels stimulates the release of human chorionic gonadotrophin and placental lactogen by placental explants. Biochim Biophys Acta1994;1220:101-106.
33.
33. Pimplikar SW, Simons K. Regulation of apical transport in epithelial cells by a Gs class of heterotrimeric G protein. Nature1993;362:456-458.
34.
34. Schultz G, Rosenthal W, Hescheler J, Trautwein W. Role of G proteins in calcium channel modulation. Annu Rev Physiol1990;52:275-292.
35.
35. Hoffmann F, Nastainczyk W, Rohrkasten A, Scheider T, Seiber M. Regulation of L-type calcium channel. Trends Pharmacol Sci1987;8:393-398.
36.
36. Collu R. Bouvier C, Lagacé G, et al. Selective deficiency of guanine nucleotide-binding protein Go in two dopamine resistant pituitary tumors. Endocrinology1988;122:1176-1178.
37.
37. Lledo PM, Vermier P, Vincent J-D, Couplage des récepteurs métabotropiques aux canaux ioniques. Rôle des protéines G. C R Soc Biol1993;1870:7-13.
38.
38. Petit A, Vaillancourt C, Bellabarba D, Lehoux J-G, Gallo-Payet N, Bélisle S. Presence of D2-dopamine receptors in human term placenta. J Recept Res1990;10:205-215.
39.
39. Vaillancourt C, Petit A, Gallo-Payet N, Bellabarba D, Lehoux J-G, Bélisle S. Labeling of D2-dopaminergic and 5-HT2-serotonergic binding sites in human trophoblastic cells using [3H]-spiperone. J Recept Res1994;14:11-22.
40.
40. Simon MI, Strathmann MP, Gautam N. Diversity of G proteins in signal transduction. Science1991;252:802-808.
41.
41. Watkins DC, Moxham CM, Morris AJ, Malbon CC. Suppression of Giα2 enhances phospholipase C signaling. Biochem J1994;299:593-596.
42.
42. Mattera R, Yatani A, Kirsch GE, et al. Recombinant alpha i-3 subunit of G protein activates Gk-gated K+ channels. J Biol Ch;em1989;264:465-471.
43.
43. Lledo PM, Homburger V, Bockaert J, Vincent J-D. Differential G proteins and Ca2+ currents in rat anterior pituitary cells. Neuron1992;8:455-463.
44.
44. Kineman RD, Getty TW, Frawley LS. Paradoxical effects of dopamine (DA): Giα3 mediates DA inhibition of PRL release while masking its PRL-releasing activity. Endocrinology1994;135:790-793.
45.
45. Ladines-Llaves CA, Maruo T, Manalo S, Mochizuki M. Cytosolic localization of epidermal growth factor and its receptor in developing placenta varies over the course of pregnancy. Am J Obstet Gynecol1991;165:1377-1382.
46.
46. Biossonnette F, Cook C, Geoghegan T, Steffen M, Henry J, Yussman MA, Schultz G. Transforming growth factor-α and epidermal growth factor messenger ribonucleic acid and protein levels in human placentas from early, mid, and late gestation. Am J Obstet Gynecol1992;166:192-199.
47.
47. Daiter E, Pampfer S, Yeung YG, Barad D, Stanley ER, Pollard JW, Expression of colony-stimulating factor-1 in the human uterus and placenta. J Clin Endocrinol Metab1992;74:850-858.
48.
48. Lysiak JJ, Han VKM, Lala PK. Localization of transforming growth factor α in the human placenta and decidua: Role in trophoblast growth. Biol Reprod1993;49:885-894.
