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Endocrine Abstracts (2022) 81 OC12.5 | DOI: 10.1530/endoabs.81.OC12.5

ECE2022 Oral Communications Oral Communications 12: Reproductive and Developmental Endocrinology (6 abstracts)

Luteinizing hormone (LH)- and choriogonadotropin (hCG)-induced internalization of the receptor (LHCGR) is responsible for hormone-specific signaling

Elia Paradiso 1 , Clara Lazzaretti 1 , Sara D’Alessandro 1,2 , Samantha Sperduti 1,3 , Neena Roy 1 , Elisa Mascolo 1 , Lara Baschieri 1,2 , Claudia Anzivino 1,3 , Manuela Simoni 1,3,4 & Livio Casarini 1,3

1University of Modena and Reggio Emilia, Unit of Endocrinology. Department of Biomedical, Metabolic and Neural Sciences, Modena, Italy; 2University of Modena and Reggio Emilia, International PhD School in Clinical and Experimental Medicine (CEM), Modena, Italy; 3University of Modena and Reggio Emilia, Center for Genomic Research, Modena, Italy; 4Azienda Ospedaliero-Universitaria di Modena, Department of Medical Specialties, Modena, Italy

Introduction: Luteinizing hormone (LH) and human choriogonadotropin (hCG) regulate reproduction through binding the same receptor (LHCGR). They act via activation of G protein- and β-arrestin-dependent signals, resulting in ligand-specific pattern of signaling cascades and LHCGR internalization into endosomal vesicles. Previous studies differentiated the action of these two hormones in LH-related proliferative signals and hCG-related steroidogenic signals.Aim. We compared the role of LHCGR internalization in determining LH- and hCG-specific signals.

Methods: Ligand-specific patterns of LHCGR trafficking and signaling were evaluated in HEK293 cells overexpressing the receptor and specific bioluminescence resonance energy transfer (BRET) biosensors, with or without internalization blockade by Dynasore. LH- and hCG-induced LHCGR internalization and trafficking were evaluated over 30 min, determining the interaction between receptor and endosomal type-specific markers RAB-GTPases (Rab) 5, 7, and 11, as well as β-arrestin 2. Ligand-specific receptor coupling to Gs, Gi, and Gq protein, and related cAMP, extracellularly-regulated kinases 1 and 2 (ERK1/2), and intracellular Ca2+ increase were evaluated as well. Results were compared by Kruskal Wallis test and Dunn’s post-test; P<0.05; n=4-8.

Results: The interaction between LHCGR and markers of internalization/localization into early endosome, i.e. β-arrestin 2 and Rab5, is markedly more induced upon cell treatment with hCG, with than LH (P<0.05; n=4). Conversely, LH induces preferential LHCGR-Rab11 interaction, indicating the routing of the receptor toward recycling in cell membrane (P<0.05; n=4), while no hormone-specific LHCGR-Rab7 interaction was found ( P≥ 0.05; n=4). Interestingly, LHCGR trafficking is modulated by the blockade of internalization with Dynasore. Under this condition, hCG-induced LHCGR-β-arrestin 2/Rab5, as well as LH-induced LHCGR-Rab11 interactions were lost ( P≥ 0.05; n=4), suggesting missing ligand-specific receptor trafficking. Moreover, Dynasore treatment increases LH-, but not hCG-induced LHCGR-Rab7 interaction, indicating ligand-specific routing toward the degradation pathway. Hormone-specific trafficking reflects the downstream signaling pattern. hCG has higher efficacy than LH in inducing Gs- and Gq coupling to LHCGR (P<0.05; n=8), reflecting more pronounced activation of cAMP and intracellular Ca2+ increase (P<0.05; n=4), as two molecules upregulating the synthesis of gonadal steroids. Cell treatment with Dynasore did neither change the hCG-related G protein coupling to LHCGR, nor cAMP production, while it decreased intracellular Ca2+ increase. Conversely, LH was more effective than hCG in inducing LHCGR coupling to Gi protein (P<0.05; n=8), preferentially activating proliferation-related ERK1/2 downstream signaling (P<0.05; n=4). However, LH-induced LHCGR-Gi coupling and ERK1/2 activation were inhibited by Dynasore (P≥ 0.05; n=4).

Conclusion: We conclude that LH-related proliferative and hCG-linked steroidogenic signals require hormone-specific trafficking of the receptor.

Volume 81

European Congress of Endocrinology 2022

Milan, Italy
21 May 2022 - 24 May 2022

European Society of Endocrinology 

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