DOI QR코드

DOI QR Code

Cell-Surface Loss of Constitutive Activating and Inactivating Mutants of Eel Luteinizing Hormone Receptors

  • Byambaragchaa, Munkhzaya (Institute of Genetic Engineering, Hankyong National University) ;
  • Choi, Seung-Hee (School of Animal Life Convergence Science, Hankyong National University) ;
  • Kim, Dong-Wan (Institute of Genetic Engineering, Hankyong National University) ;
  • Min, Kwan-Sik (Institute of Genetic Engineering, Hankyong National University)
  • 투고 : 2021.08.21
  • 심사 : 2021.12.11
  • 발행 : 2021.12.31

초록

The present study aimed to investigate the mechanism of cell surface receptor loss by two constitutively activating mutants (designated L469R, and D590Y) and two inactivating mutants (D417N and Y558F) of the luteinizing hormone receptor (LHR) in the Japanese eel Anguilla japonica, known to naturally occur in human LHR transmembrane domains. We investigated cell surface receptor loss using an enzyme-linked immunosorbent assay in HEK 293 cells. The expression level of wild-type eel LHR was considered to be 100%, and the expression levels of L469R and D417N were 97% and 101%, respectively, whereas the expression levels of D590Y and Y558F slightly increased to approximately 110% and 106%, respectively. The constitutively activating mutants L469R and D590Y exhibited a decrease in cell surface loss in a manner similar to that of wild-type eel LHR. The rates of loss of cell surface agonist-receptor complexes were observed to be very rapid (2.6-6.2 min) in both the wild-type eel LHR and activating mutants. However, cell surface receptor loss in the cells expressing inactivating mutants D417N and Y558F was slightly observed in the cells expressing inactivating mutants D417N and Y558F, despite treatment with a high concentration of agonist. These results provide important information on LHR function in fish and the regulation of mutations of highly conserved amino acids in glycoprotein hormone receptors.

키워드

참고문헌

  1. Ascoli M, Fanelli F, Segaloff DL (2002) The lutropin/choriogonadotropin receptor, a 2002 perspective. Endocr Rev 23:141-174. https://doi.org/10.1210/er.23.2.141
  2. Bhaskaran RS, Ascoli M (2005) The post-endocytotic fate of the gonadotropin receptors is an important determinant of the desensitization of gonadotropin responses. J Mol Endocrinol 34:447-457. https://doi.org/10.1677/jme.1.01745
  3. Byambaragchaa M, Kim DJ, Kang MH, Min KS (2018a) Site specificity of eel luteinizing hormone N-linked oligosaccharides in signal transduction. Gen Comp Endocrinol 268:50-56. https://doi.org/10.1016/j.ygcen.2018.07.015
  4. Byambaragchaa M, Lee SY, Kim DJ, Kang MH, Min KS (2018b) Signal transduction of eel luteinizing hormone receptor (eelLHR) and follicle stimulating hormone receptor (eelFSHR) by recombinant equine chorionic gonadotropin (rec-eCG) and native eCG. Dev Reprod 22:55-64. https://doi.org/10.12717/DR.2018.22.1.055
  5. Byambaragchaa M, Kim JS, Park HK, Kim DJ, Hong SM, Kang MH, Min KS (2020) Constitutive activation and inactivation of mutations inducing cell surface loss of receptor and impairing of signal transduction of agonist-stimulated eel follicle-stimulating hormone receptor. Int J Mol Sci 21:7075. https://doi.org/10.3390/ijms21197075
  6. Byambaragchaa M, Park A, Gil SJ, Lee HW, Ko YJ, Choi SH, Kang MH, Min KS (2021a) Luteinizing hormone-like and follicle-stimulating hormone-like activities of equine chorionic gonadotropin β-subunit mutants in cells expressing rat luteinizing hormone/chorionic gonadotropin receptor and rat follicle-stimulating hormone receptor. Anim Cells Syst 25:171-181. https://doi.org/10.1080/19768354.2021.1943708
  7. Byambaragchaa M, Seong HK, Choi SH, Kim DJ, Kang MH, Min KS (2021b) Constitutively activating mutants of equine LH/CGR constitutively induce signal transduction and inactivating mutations impair biological activity and cell-surface receptor loss in vitro. Int J Mol Sci 22:10723. https://doi.org/10.3390/ijms221910723
  8. Byambaragchaa M, Choi SH, Kim DW, Min KS (2021c) Constitutive activating eel luteinizing hormone receptors induce constitutively signal transduction and inactivating mutants impair biological activity. Dev Reprod 25:133-143. https://doi.org/10.12717/DR.2021.25.3.133
  9. Byambaragchaa M, Ahn TY, Choi SH, Kang MH, Min KS (2021d) Functional characterization of naturally-occurring constitutively activating/inactivating mutations in equine follicle-stimulating hormone receptor (eFSHR). Anim Biosci.
  10. Dhanwada KR, Vijapurkar U, Ascoli M (1996) Two mutations of the lutropin/choriogonadotropin receptor that impair signal transduction also interfere with receptor-mediated endocytosis. Mol Endocrinol 10:544-554. https://doi.org/10.1210/mend.10.5.8732685
  11. Foster SR, Brauner-Osborne H (2018) Investigating internalization and intracellular trafficking of GPCRs: New techniques and real-time experimental approaches. In: Ulloa-Aguirre A, Tao YX (ed), Targeting trafficking in drug development. Handbook of Experimental Pharmacology. Springer, Cham.
  12. Galet C, Ascoli M (2006) A constitutively active mutant of the human lutropin receptor (hLHR-L457R) escapes lysosomal targeting and degradation. Mol Endocrinol 20:2931-2945. https://doi.org/10.1210/me.2006-0138
  13. Jacobsen SE, Ammendrup-Johnsen I, Jansen AM, Gether U, Madsen KL, Brauner-Osborne H (2017) The GPRC6A receptor displays constitutive internalization and sorting to the slow recycling pathway. J Biol Chem 292:6910-6926. https://doi.org/10.1074/jbc.M116.762385
  14. Kim DJ, Park CW, Kim DW, Park HK, Byambaragchaa M, Lee NS, Hong SM, Seo MY, Kang MH, Min KS (2016) Production and characterization of monoclonal antibodies against recombinant tethered follicle-stimulating hormone from Japanese eel Anguilla japonica. Gen Comp Endocrinol 233:8-15. https://doi.org/10.1016/j.ygcen.2016.04.030
  15. Kim JM, Byambaragchaa M, Kang MH, Min KS (2018) The C-terminal phosphorylation sites of eel follicle-stimulating hormone receptor are important role in the signal transduction. Dev Reprod 22:143-153. https://doi.org/10.12717/DR.2018.22.2.143
  16. Kim JM, Munkhuu O, Byambaragchaa M, Lee BI, Kim SK, Kang MH, Min KS (2019) Site-specific roles of N-linked oligosaccharides in recombinant eel follicle-stimulating hormone for secretion and signal transduction. Gen Comp Endocrinol 276:37-44. https://doi.org/10.1016/j.ygcen.2019.03.003
  17. Kudo M, Osuga Y, Kobilka BK, Hsueh AJW (1996) Transmembrane regions V and VI of the human luteinizing hormone receptor are required for constitutive activation by a mutation in the third intracellular loop. J Biol Chem 271:22470-22478. https://doi.org/10.1074/jbc.271.37.22470
  18. Latronico AC, Chai Y, Arnhold IJP, Liu X, Mendonca BB, Segaloff DL (1998) A homozygous microdeletion in helix 7 of the luteinizing hormone receptor associated with familial testicular and ovarian resistance is due to both decreased cell surface expression and impaired effector activation by the cell surface receptor. Mol Endocrinol 12:442-450. https://doi.org/10.1210/mend.12.3.0077
  19. Latronico AC, Segaloff DL (2007) Insights learned from L457(3.43)R, an activating mutant of the human lutropin receptor. Mol Cell Endocrinol 260-262:287-293. https://doi.org/10.1016/j.mce.2005.11.053
  20. Lee SY, Byambaragchaa M, Choi SH, Kang HJ, Kang MH, Min KS (2021) Roles of N-linked and O-linked glycosylation sites in the activity of equine chorionic gonadotropin in cells expressing rat luteinizing hormone/chorionic gonadotropin receptor and follicle-stimulating hormone receptor. BMC Biotechnol 21:52. https://doi.org/10.1186/s12896-021-00712-8
  21. McGee SR, Narayan P (2013) Precocious puberty and Leydig cell hyperplasia in male mice with a gain of function mutation in the LH receptor gene. Endocrinology 154:3900-3913. https://doi.org/10.1210/en.2012-2179
  22. Meehan TP, Narayan P (2007) Constitutively active luteinizing hormone receptors: Consequences of in vivo expression. Mol Cell Endocrinol 260-262:294-300. https://doi.org/10.1016/j.mce.2006.03.045
  23. Min KS, Liu X, Fabritz J, Jaquette J, Abell AN, Ascoli M (1998) Mutations that induce constitutive activation and mutations that impair signal transduction modulate the basal and/or agonist-stimulated internalization of the lutropin/choriogonadotropin receptor. J Biol Chem 273:34911-34919. https://doi.org/10.1074/jbc.273.52.34911
  24. Mundell SJ, Matharu AL, Nisar S, Palmer TM, Benovic JL, Kelly E (2010) Deletion of the distal COOH-terminus of the A2B adenosine receptor switches internalization to an arrestin- and clathrin-independent pathway and inhibits recycling. Br J Pharmacol 159:518-533. https://doi.org/10.1111/j.1476-5381.2009.00598.x
  25. Shenker A, Laue L, Kosugi S, Merendino Jr. JJ, Minegishi T, Cutler Jr. GB (1993) A constitutively activating mutation of the luteinizing hormone receptor in familial male precocious puberty. Nature 365:652-654. https://doi.org/10.1038/365652a0
  26. Simoni M, Gromoll J, Nieschlag E (1997) The follicle-stimulating hormone receptor: Biochemistry, molecular biology, physiology, and pathophysiology. Endocr Rev 18:739-773. https://doi.org/10.1210/er.18.6.739
  27. Tao YX (2006) Inactivating mutations of G protein-coupled receptors and diseases: Structurefunction insights and therapeutic implications. Pharmacol Ther 111:949-973. https://doi.org/10.1016/j.pharmthera.2006.02.008
  28. Tao YX, Abell AN, Liu X, Nakamura K, Segaloff DL (2000) Constitutive activation of G protein-coupled receptors as a result of selective substitution of a conserved leucine residue in transmembrane helix III. Mol Endocrinol 14:1272-1282. https://doi.org/10.1210/mend.14.8.0503
  29. Themmen APN, Huhtaniemi IT (2000) Mutations of gonadotropins and gonadotropin receptors: Elucidating the physiology and pathophysiology of pituitary-gonadal function. Endocr Rev 21:551-583. https://doi.org/10.1210/er.21.5.551
  30. Yano K, Kohn LD, Saji M, Kataoka N, Okuno A, Cutler Jr. GB (1996) A case of male-limited precocious puberty caused by a point mutation in the second transmembrane domain of the luteinizing hormone choriogonadotropin receptor gene. Biochem Biophys Res Commun 220:1036-1042. https://doi.org/10.1006/bbrc.1996.0528
  31. Zhang M, Mizrachi D, Fanelli F, Segaloff DL (2005) The formation of a salt bridge between helices 3 and 6 is responsible for the constitutive activity and lack of hormone responsiveness of the naturally occurring L457R mutation of the human lutropin receptor. J Biol Chem 280:26169-26176. https://doi.org/10.1074/jbc.M502102200
  32. Zhang M, Tao YX, Ryan GL, Feng X, Fanelli F, Segaloff DL (2007) Intrinsic differences in the response of the human lutropin receptor versus the human follitropin receptor to activating mutations. J Biol Chem 282:25527-25539. https://doi.org/10.1074/jbc.M703500200