Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

Constitutive Activating and Inactivating Mutants of Eel Luteinizing Hormone Receptor

Version 1 : Received: 6 September 2020 / Approved: 8 September 2020 / Online: 8 September 2020 (10:56:48 CEST)

How to cite: Byambaragchaa, M.; Kim, D.; Kim, D.; Hong, S.; Kang, M.; Min, K. Constitutive Activating and Inactivating Mutants of Eel Luteinizing Hormone Receptor. Preprints 2020, 2020090185 (doi: 10.20944/preprints202009.0185.v1). Byambaragchaa, M.; Kim, D.; Kim, D.; Hong, S.; Kang, M.; Min, K. Constitutive Activating and Inactivating Mutants of Eel Luteinizing Hormone Receptor. Preprints 2020, 2020090185 (doi: 10.20944/preprints202009.0185.v1).

Abstract

We analyzed signal transduction of three constitutively activating mutants (M410T, L469R, and D590Y) and two inactivating mutants (D417N and Y558F) of the eel luteinizing hormone receptor (eel LHR), known to occur in human LHR. The objective of this study was to assess the functional effects of these mutations in signal transduction and cell surface loss of receptor. Mutant receptors were transiently expressed in Chinese hamster ovary (CHO-K1) cells. Eel LH-stimulated accumulation of cyclic adenosine monophosphate (cAMP) was measured by homogeneous time-resolved fluorescence (HTRF) assays. The loss of receptors from the cells surface was measured using an enzyme-linked immunosorbent assay (ELISA) in human embryonic kidney (HEK) 293 cells. The cAMP response in cells expressing the wild type eel LHR was increased in a dose-dependent manner using eel LH ligand stimulation. Compared with the wild type, cells expressing the activating mutants (M410T, L469R, and D590Y), exhibited a 4.0-, 19.1-, and 7.8-fold increase in basal cAMP response without agonist stimulation, respectively. Their maximal responses to agonist stimulation were approximately 65%, 52%, and 98%, respectively, of those of the wild type. The inactivating mutants (D417N and Y558F) did not completely impair signal transduction, and their maximal responses were only 33% and25 % of those of wild type. These data clearly showed that the eel LHR-L469R and D590Y, activating mutants enhanced the rate of the loss of cell surface receptors following treatment with eel LH. Thus, the loss of cell surface receptors in cells expressing mutant eel LHRs was consistent with the eel LH agonist-induced production of cAMP. Our results suggested that the activation of the eel LHR requires appropriate loss of LHR-ligand complexes from the cell surface.

Subject Areas

eel luteinizing hormone receptor; constitutively activating mutation; inactivating mutation; cyclic adenosine monophosphate response; cell surface loss of receptor

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