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Heterologous, PKC-Mediated Desensitization of Human Histamine H3 Receptors Expressed in CHO-K1 Cells

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Abstract

Desensitization is a major mechanism to regulate the functional response of G protein-coupled receptors. In this work we studied whether the human histamine H3 receptor of 445 amino acids (hH3R445) experiences heterologous desensitization mediated by PKC activation. Bioinformatic analysis indicated the presence of Serine and Threonine residues susceptible of PKC-mediated phosphorylation on the third intracellular loop and the carboxyl terminus of the hH3R445. In CHO-K1 cells stably transfected with the hH3R445 direct PKC activation by phorbol 12-myristate 13-acetate (TPA, 200 nM) abolished H3R-mediated inhibition of forskolin-stimulated cAMP accumulation. Activation of endogenous purinergic receptors by ATP (adenosine 5′-triphosphate, 10 μM) increased the free calcium intracellular concentration ([Ca2+]i) confirming their coupling to phospholipase C stimulation. Incubation with ATP also abolished H3R-mediated inhibition of forskolin-induced cAMP accumulation, and this effect was prevented by the PKC inhibitors Ro-31-8220 and Gö-6976. Pre-incubation with TPA or ATP reduced H3R-mediated stimulation of [35S]-GTPγS binding to membranes from CHO-K1-hH3R445 cells by 39.7 and 54.2 %, respectively, with no change in the agonist potency, and the effect was prevented by either Ro-31-8220 or Gö-6976. Exposure to ATP or TPA also resulted in the loss of cell surface H3Rs (−30.4 and −45.1 %) as evaluated by [3H]-NMHA binding to intact cells. These results indicate that the hH3R445 undergoes heterologous desensitization upon activation of receptors coupled to PKC stimulation.

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Abbreviations

AC:

Adenylyl cyclase

DAG:

Diacylglycerol

H3R:

Histamine H3 receptor

GPCR:

G protein-coupled receptor

GRK:

GPCR kinase

PLC:

Phospholipase C

PKC:

Protein kinase C

RAMH:

R-α-methylhistamine

TPA:

Phorbol 12-myristate 13-acetate

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Acknowledgments

Supported by Cinvestav and Conacyt (Grant 128205 to J.-A.A.-M.). We thank Raul Gonzalez-Pantoja for excellent technical assistance. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The funding sources were not involved at all in the study design, collection, analysis and interpretation of data, writing of the manuscript or the decision to submit this report.

Author Contributions

W. M.-L., U. G.-H. and J.-A.A.-M. designed the study; W. M.-L., N. R.-R. and J. E.-S. conducted experiments; W. M.-L., N. R.-R., U. G.-H. and J.-A.A.-M. performed data analysis. W. M.-L., U. G.-H. and J.-A.A.-M. wrote the manuscript. All authors revised and approved the manuscript.

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Authors and Affiliations

  1. Departamento de Fisiología, Biofísica y Neurociencias, Centro de Investigación y de Estudios Avanzados del IPN, Av. IPN 2508, Zacatenco, 07360, México, D.F., Mexico

    Wilber Montejo-López, Nayeli Rivera-Ramírez, Juan Escamilla-Sánchez, Ubaldo García-Hernández & José-Antonio Arias-Montaño

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  1. Wilber Montejo-López
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  2. Nayeli Rivera-Ramírez
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  3. Juan Escamilla-Sánchez
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Correspondence to José-Antonio Arias-Montaño.

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Montejo-López, W., Rivera-Ramírez, N., Escamilla-Sánchez, J. et al. Heterologous, PKC-Mediated Desensitization of Human Histamine H3 Receptors Expressed in CHO-K1 Cells. Neurochem Res 41, 2415–2424 (2016). https://doi.org/10.1007/s11064-016-1954-5

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  • DOI: https://doi.org/10.1007/s11064-016-1954-5

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