Preprint Article Version 1 This version is not peer-reviewed

Mutations in Conserved Regions of Eel Luteinizing Hormone Receptor Result in Constitutive Activation or Inactivation

Version 1 : Received: 4 July 2019 / Approved: 8 July 2019 / Online: 8 July 2019 (12:38:11 CEST)

How to cite: Byambaragchaa, M.; Kim, D.; Kim, D.; Hong, S.; Kang, M.; Min, K. Mutations in Conserved Regions of Eel Luteinizing Hormone Receptor Result in Constitutive Activation or Inactivation. Preprints 2019, 2019070117 (doi: 10.20944/preprints201907.0117.v1). Byambaragchaa, M.; Kim, D.; Kim, D.; Hong, S.; Kang, M.; Min, K. Mutations in Conserved Regions of Eel Luteinizing Hormone Receptor Result in Constitutive Activation or Inactivation. Preprints 2019, 2019070117 (doi: 10.20944/preprints201907.0117.v1).

Abstract

Luteinizing hormone receptor (LHR) is a member of the seven-transmembrane (TM) receptor family. Several mutations in LHR have been identified in many mammalian species, leading to either constitutive activation or inactivation of the receptor. Mutations in highly conserved regions of the TM domain have been reported. In this study, we analyzed signal transduction by three constitutively active mutants (designated M410T, L469R, and D590Y) and two inactivating mutants (D383N and Y546F) of eelLHR known as naturally occurring in human LHR . To directly assess the functional effects of these mutations, site-directed mutant receptors were transiently expressed in CHO-K1 cells and cAMP accumulation stimulated by recombinant eelLH (rec-eelLH) was measured by homogeneous time-resolved fluorescence (HTRF) assays. The cAMP response in cells expressing eelLHR wild-type (eLHR-WT) increased in a dose-dependent manner with rec-eelLH ligand stimulation. Cells expressing the activating eelLHR mutants, M410T, L469R, and D590Y, exhibited a 4.0-, 19.1-, and 7.8-fold increase in basal cAMP response, respectively. However, their maximal responses were approximately 73, 53, and 92%, respectively, of the maximal response of LHR-WT. The L469R mutant exhibited a particularly marked increase in cAMP production in the absence of agonist. The maximal responses of the inactivating mutants, D383N and Y546F, were 32 and 24% of LHR-WT, respectively. However, the inactivating mutations did not completely impair signal transduction. Thus, we report here the first characterization of activating and inactivating mutations in eelLHR and we show that these mutations have similar effects as those reported for mammalian LHRs. Moreover, eelLHR with activating mutations showed constitutive cAMP responses. These results provide important data on the signal transduction of constitutively active and inactive LHR mutants. Further studies should aim to identify the mechanism responsible for the significant increase in basal cAMP response in the constitutively activated eelLHR mutants.

Subject Areas

eel luteinizing hormone receptor, constitutively activating mutation, inactivating mutation, cAMP responses.

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