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1.
Int J Mol Sci ; 25(14)2024 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-39062808

RESUMO

The melanocortin-4 receptor (MC4R) is a key player in the hypothalamic leptin-melanocortin pathway that regulates satiety and hunger. MC4R belongs to the G protein-coupled receptors (GPCRs), which are known to form heterodimers with other membrane proteins, potentially modulating receptor function or characteristics. Like MC4R, thyroid hormones (TH) are also essential for energy homeostasis control. TH transport across membranes is facilitated by the monocarboxylate transporter 8 (MCT8), which is also known to form heterodimers with GPCRs. Based on the finding in single-cell RNA-sequencing data that both proteins are simultaneously expressed in hypothalamic neurons, we investigated a putative interplay between MC4R and MCT8. We developed a novel staining protocol utilizing a fluorophore-labeled MC4R ligand and demonstrated a co-localization of MC4R and MCT8 in human brain tissue. Using in vitro assays such as BRET, IP1, and cAMP determination, we found that MCT8 modulates MC4R-mediated phospholipase C activation but not cAMP formation via a direct interaction, an effect that does not require a functional MCT8 as it was not altered by a specific MCT8 inhibitor. This suggests an extended functional spectrum of MCT8 as a GPCR signaling modulator and argues for the investigation of further GPCR-protein interactions with hitherto underrepresented physiological functions.


Assuntos
Transportadores de Ácidos Monocarboxílicos , Receptor Tipo 4 de Melanocortina , Fosfolipases Tipo C , Humanos , Receptor Tipo 4 de Melanocortina/metabolismo , Receptor Tipo 4 de Melanocortina/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Fosfolipases Tipo C/metabolismo , Células HEK293 , Transdução de Sinais , AMP Cíclico/metabolismo , Simportadores/metabolismo , Simportadores/genética , Ligação Proteica , Animais
2.
Int J Mol Sci ; 25(4)2024 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-38397010

RESUMO

A wound healing model was developed to elucidate the role of mesenchymal-matrix-associated transglutaminase 2 (TG2) in keratinocyte re-epithelialisation. TG2 drives keratinocyte migratory responses by activation of disintegrin and metalloproteinase 17 (ADAM17). We demonstrate that epidermal growth factor (EGF) receptor ligand shedding leads to EGFR-transactivation and subsequent rapid keratinocyte migration on TG2-positive ECM. In contrast, keratinocyte migration was impaired in TG2 null conditions. We show that keratinocytes express the adhesion G-protein-coupled receptor, ADGRG1 (GPR56), which has been proposed as a TG2 receptor. Using ADAM17 activation as a readout and luciferase reporter assays, we demonstrate that TG2 activates GPR56. GPR56 activation by TG2 reached the same level as observed with an agonistic N-GPR56 antibody. The N-terminal GPR56 domain is required for TG2-regulated signalling response, as the constitutively active C-GPR56 receptor was not activated by TG2. Signalling required the C-terminal TG2 ß-barrel domains and involved RhoA-associated protein kinase (ROCK) and ADAM17 activation, which was blocked by specific inhibitors. Cell surface binding of TG2 to the N-terminal GPR56 domain is rapid and is associated with TG2 and GPR56 endocytosis. TG2 and GPR56 represent a ligand receptor pair causing RhoA and EGFR transactivation. Furthermore, we determined a binding constant for the interaction of human TG2 with N-GPR56 and show for the first time that only the calcium-enabled "open" TG2 conformation associates with N-GPR56.


Assuntos
Proteína 2 Glutamina gama-Glutamiltransferase , Receptores Acoplados a Proteínas G , Humanos , Proteína ADAM17/genética , Proteína ADAM17/metabolismo , Receptores ErbB/metabolismo , Ligantes , Proteína 2 Glutamina gama-Glutamiltransferase/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais
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