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1.
J Biol Chem ; 296: 100163, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33288675

RESUMO

Uveal melanoma is the most common eye cancer in adults and is clinically and genetically distinct from skin cutaneous melanoma. In a subset of cases, the oncogenic driver is an activating mutation in CYSLTR2, the gene encoding the G protein-coupled receptor cysteinyl-leukotriene receptor 2 (CysLTR2). The mutant CYSLTR2 encodes for the CysLTR2-L129Q receptor, with the substitution of Leu to Gln at position 129 (3.43). The ability of CysLTR2-L129Q to cause malignant transformation has been hypothesized to result from constitutive activity, but how the receptor could escape desensitization is unknown. Here, we characterize the functional properties of CysLTR2-L129Q. We show that CysLTR2-L129Q is a constitutively active mutant that strongly drives Gq/11 signaling pathways. However, CysLTR2-L129Q only poorly recruits ß-arrestin. Using a modified Slack-Hall operational model, we quantified the constitutive activity for both pathways and conclude that CysLTR2-L129Q displays profound signaling bias for Gq/11 signaling pathways while escaping ß-arrestin-mediated downregulation. CYSLTR2 is the first known example of a G protein-coupled receptor driver oncogene that encodes a highly biased constitutively active mutant receptor. These results provide new insights into the mechanism of CysLTR2-L129Q oncoprotein signaling and suggest CYSLTR2 as a promising potential therapeutic target in uveal melanoma.


Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Regulação Neoplásica da Expressão Gênica , Receptores de Leucotrienos/genética , Transdução de Sinais/genética , beta-Arrestina 2/genética , Substituição de Aminoácidos , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Glutamina/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Cinética , Lisina/metabolismo , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Modelos Biológicos , Mutação , Ligação Proteica , Receptores de Leucotrienos/metabolismo , Receptores de Vasopressinas/genética , Receptores de Vasopressinas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Neoplasias Uveais/genética , Neoplasias Uveais/metabolismo , Neoplasias Uveais/patologia , beta-Arrestina 2/metabolismo
2.
Cell Mol Neurobiol ; 41(5): 1085-1101, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33216235

RESUMO

Many G protein-coupled receptors (GPCRs) signal through more than one subtype of heterotrimeric G proteins. For example, the C-C chemokine receptor type 5 (CCR5), which serves as a co-receptor to facilitate cellular entry of human immunodeficiency virus 1 (HIV-1), normally signals through the heterotrimeric G protein, Gi. However, CCR5 also exhibits G protein signaling bias and certain chemokine analogs can cause a switch to Gq pathways to induce Ca2+ signaling. We want to understand how much of the Ca2+ signaling from Gi-coupled receptors is due to G protein promiscuity and how much is due to transactivation and crosstalk with other receptors. We propose a possible mechanism underlying the apparent switching between different G protein signaling pathways. We show that chemokine-mediated Ca2+ flux in HEK293T cells expressing CCR5 can be primed and enhanced by ATP pretreatment. In addition, agonist-dependent lysosomal exocytosis results in the release of ATP to the extracellular milieu, which amplifies cellular signaling networks. ATP is quickly degraded via ADP and AMP to adenosine. ATP, ADP and adenosine activate different cell surface purinergic receptors. Endogenous Gq-coupled purinergic P2Y receptors amplify Ca2+ signaling and allow for Gi- and Gq-coupled receptor signaling pathways to converge. Associated secretory release of GPCR ligands, such as chemokines, opioids, and monoamines, should also lead to concomitant release of ATP with a synergistic effect on Ca2+ signaling. Our results suggest that crosstalk between ATP-activated purinergic receptors and other Gi-coupled GPCRs is an important cooperative mechanism to amplify the intracellular Ca2+ signaling response.


Assuntos
Sinalização do Cálcio/fisiologia , Receptor Cross-Talk/fisiologia , Receptores CCR5/agonistas , Receptores CCR5/metabolismo , Receptores Purinérgicos/metabolismo , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Quimiocina CCL5/metabolismo , Quimiocina CCL5/farmacologia , Células HEK293 , Humanos , Agonistas Purinérgicos/metabolismo , Agonistas Purinérgicos/farmacologia , Antagonistas Purinérgicos/metabolismo , Antagonistas Purinérgicos/farmacologia , Receptor Cross-Talk/efeitos dos fármacos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/metabolismo , Suramina/metabolismo , Suramina/farmacologia
3.
Cell Chem Biol ; 27(6): 642-644, 2020 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-32559501

RESUMO

In this issue of Cell Chemical Biology, White et al. (2020) describe CRISPR/Cas9-mediated tagging of GPCRs and ß-arrestin to provide a method to study receptor signaling in cells under conditions of endogenous genetic control. The strategy, when coupled with luminescence reporter and complementation technologies, provides new avenues to study GPCRs.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Receptores de Quimiocinas , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , beta-Arrestinas
4.
Biophys J ; 117(5): 903-919, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31421836

RESUMO

The chemokine receptor CCR5 is a drug target to prevent transmission of HIV/AIDS. We studied four analogs of the native chemokine regulated, on activation, normal T-cell-expressed, and secreted (RANTES) (CCL5) that have anti-HIV potencies of around 25 pM, which is more than four orders of magnitude higher than that of RANTES itself. It has been hypothesized that the ultrahigh potency of the analogs is due to their ability to bind populations of receptors not accessible to native chemokines. To test this hypothesis, we developed a homogeneous dual-color fluorescence cross-correlation spectroscopy assay for saturation- and competition-binding experiments. The fluorescence cross-correlation spectroscopy assay has the advantage that it does not rely on competition with radioactively labeled native chemokines used in conventional assays. We prepared site-specifically labeled fluorescent analogs using native chemical ligation of synthetic peptides, followed by bioorthogonal fluorescent labeling. We engineered a mammalian cell expression construct to provide fluorescently labeled CCR5, which was purified using a tandem immunoaffinity and size-exclusion chromatography approach to obtain monomeric fluorescent CCR5 in detergent solution. We found subnanomolar binding affinities for the two analogs 5P12-RANTES and 5P14-RANTES and about 20-fold reduced affinities for PSC-RANTES and 6P4-RANTES. Using homologous and heterologous competition experiments with unlabeled chemokine analogs, we conclude that the analogs all bind at the same binding site, whereas the native chemokines (RANTES and MIP-1α) fail to displace bound fluorescent analogs even at tens of micromolar concentrations. Our results can be rationalized with de novo structural models of the N-terminal tails of the synthetic chemokines that adopt a different binding mode as compared to the parent compound.


Assuntos
Quimiocinas/metabolismo , HIV-1/metabolismo , Receptores CCR5/metabolismo , Ligação Competitiva , Quimiocina CCL5/metabolismo , Células HEK293 , Humanos , Ligantes , Modelos Biológicos , Ligação Proteica
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