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A single-point mutation (Ala280Val) in the third intracellular loop alters the signalling properties of the human histamine H? receptor stably expressed in CHO-K1 cells.

ABSTRACT: BACKGROUND AND PURPOSE: An alanine to valine exchange at amino acid position 280 (A280V) in the third intracellular loop of the human histamine H? receptor was first identified in a patient suffering from Shy-Drager syndrome and later reported as a risk factor for migraine. Here, we have compared the pharmacological and signalling properties of wild-type (hH? R(WT)) and A280V mutant (hH? R(A280V)) receptors stably expressed in CHO-K1 cells. EXPERIMENTAL APPROACH: The hH? R(A280V) cDNA was created by overlapping extension PCR amplification. Receptor expression and affinity were assessed by radioligand (N-?-[methyl-³H]-histamine) binding to cell membranes, and receptor function by the inhibition of forskolin-induced cAMP accumulation and stimulation of ERK1/2 phosphorylation in intact cells, as well as stimulation of [³?S]-GTP?S binding to cell membranes. KEY RESULTS: Both receptors were expressed at similar levels with no significant differences in their affinities for H? receptor ligands. Upon activation the hH? RWT was significantly more efficacious to inhibit forskolin-induced cAMP accumulation and to stimulate [³?S]-GTP?S binding, with no difference in pEC50 estimates. The hH? RWT was also more efficacious to stimulate ERK1/2 phosphorylation, but this difference was not significant. The inverse agonist ciproxifan was more efficacious at hH3 RWT to reduce [³?S]-GTP?S binding but, for both receptors, failed to enhance forskolin-induced cAMP accumulation. CONCLUSIONS AND IMPLICATIONS: The A280V mutation reduces the signalling efficacy of the human H? receptor. This effect may be relevant to the pathophysiology of disorders associated with the mutation.

SUBMITTER: Flores-Clemente C

PROVIDER: S-EPMC3764854 | biostudies-other | 2013 Sep

REPOSITORIES: biostudies-other

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A single-point mutation (Ala280Val) in the third intracellular loop alters the signalling properties of the human histamine H₃ receptor stably expressed in CHO-K1 cells.

Flores-Clemente Cecilia C Osorio-Espinoza Angélica A Escamilla-Sánchez Juan J Leurs Rob R Arias Juan-Manuel JM Arias-Montaño José-Antonio JA

British journal of pharmacology 20130901 1

<h4>Background and purpose</h4>An alanine to valine exchange at amino acid position 280 (A280V) in the third intracellular loop of the human histamine H₃ receptor was first identified in a patient suffering from Shy-Drager syndrome and later reported as a risk factor for migraine. Here, we have compared the pharmacological and signalling properties of wild-type (hH₃ R(WT)) and A280V mutant (hH₃ R(A280V)) receptors stably expressed in CHO-K1 cells.<h4>Experimental approach</h4>The hH₃ R(A280V) cD ...[more]

PMID: 23713487

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Project description:BackgroundTo ensure maximal productivity of recombinant proteins (rP) during production culture it is typical to encourage an initial phase of rapid cell proliferation to achieve high biomass followed by a stationary phase where cellular energies are directed towards production of rP. During many such biphasic cultures, the initial phase of rapid cell growth at 37 degrees C is followed by a growth arrest phase induced through reduction of the culture temperature. Low temperature induced growth arrest is associated with many positive phenotypes including increased productivity, sustained viability and an extended production phase, although the mechanisms regulating these phenotypes during mild hypothermia are poorly understood.ResultsIn this study differential protein expression in suspension CHO-K1 cells was investigated following a reduction of the culture temperature from 37 degrees C to 31 degrees C in comparison to standard batch culture maintained at 37 degrees C using 2D-DIGE (Fluorescence 2-D Difference Gel Electrophoresis) and mass spectrometry (MS). There is only limited proteomic analysis of suspension-grown CHO cells describing a direct comparison of temperature shifted versus non-temperature shifted cultures using 2D-DIGE. This investigation has enabled the identification of temperature-dependent as well as temperature-independent proteomic changes. 201 proteins were observed as differentially expressed following temperature shift, of which 118 were up regulated. Of the 53 proteins identified by MALDI-ToF MS, 23 were specifically differentially expressed upon reduction of the culture temperature and were found related to a variety of cellular functions such as regulation of growth (HNRPC), cap-independent translation (EIF4A), apoptosis (importin-alpha), the cytoskeleton (vimentin) and glycoprotein quality control (alpha glucosidase 2).ConclusionThese results indicate the extent of the temperature response in CHO-K1 cells and suggest a number of key regulatory proteins and pathways that are involved in modulating the response of cells to mild hypothermia. Regulation of these identified proteins and pathways could be useful for future approaches to engineer CHO cells for improved recombinant protein production.

Dataset Information

A single-point mutation (Ala280Val) in the third intracellular loop alters the signalling properties of the human histamine H? receptor stably expressed in CHO-K1 cells.

Publications

A single-point mutation (Ala280Val) in the third intracellular loop alters the signalling properties of the human histamine H₃ receptor stably expressed in CHO-K1 cells.

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