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Distinct expression and ligand-binding profiles of two constitutively active GPR17 splice variants.

ABSTRACT: BACKGROUND AND PURPOSE: In humans and non-human primates, the 7TM receptor GPR17 exists in two isoforms differing only by the length of the N-terminus. Of these, only the short isoform has previously been characterized. Hence, we investigated gene expression and ligand-binding profiles of both splice variants and furthermore uncovered and characterized constitutive activity of both isoforms. EXPERIMENTAL APPROACH: Expression levels of the hGPR17 isoforms were determined in several brain regions as well as heart and kidney using quantitative RT-PCR. A CREB reporter assay and [(35)S]-GTPgammaS binding were employed to assess the constitutive activity and the activation by UDP, UDP-glucose and -galactose and the cysteinyl leukotrienes LTC(4) and LTD(4). Leukotriene binding and induction of internalization were furthermore tested using homologous competition binding and antibody-feeding experiments respectively. KEY RESULTS: The short isoform (hGPR17-S) was expressed more abundantly (eight- to 23-fold) in the brain than the long isoform (hGPR17-L), whereas the opposite was observed in heart and kidney. As previously reported, the uracil nucleotides activated hGPR17-S with micromolar potencies. However, much lower potencies were observed for hGPR17-L with a 50- to 170-fold increase in EC(50). Furthermore, contrary to previous reports, neither of the isoforms was activated or bound by the cysteinyl leukotrienes. Finally, both receptors were demonstrated to be constitutively active through Galpha(i). CONCLUSIONS AND IMPLICATIONS: We present the first isoform-specific characterization of GPR17 and show that differences exist between the isoforms, in both expression pattern and pharmacological profile. In turn, our results indicate that the two human isoforms might serve tissue-specific functions.

SUBMITTER: Benned-Jensen T

PROVIDER: S-EPMC2839267 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Distinct expression and ligand-binding profiles of two constitutively active GPR17 splice variants.

Benned-Jensen T T Rosenkilde M M MM

British journal of pharmacology 20100208 5

<h4>Background and purpose</h4>In humans and non-human primates, the 7TM receptor GPR17 exists in two isoforms differing only by the length of the N-terminus. Of these, only the short isoform has previously been characterized. Hence, we investigated gene expression and ligand-binding profiles of both splice variants and furthermore uncovered and characterized constitutive activity of both isoforms.<h4>Experimental approach</h4>Expression levels of the hGPR17 isoforms were determined in several b ...[more]

PMID: 20148890

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Project description:BackgroundStat5 is a latent transcription factor that regulates essential growth and survival functions in normal cells. Constitutive activity of Stat5 and the involvement of its C-terminally truncated variant have been implicated in blood cell malignancies and mammary or breast cancer. To distinguish the individual contributions of the Stat5 variants to mammary tumorigenesis, global gene-expression profiling was performed on transgenic STAT5-induced tumors.ResultsWe identified 364 genes exhibiting differential expression in mammary tumors developed in transgenic mice expressing constitutively active STAT5 (STAT5ca) vs. its C-terminally truncated variant (STAT5Delta750). These genes mediate established Stat5 effects on cellular processes such as proliferation and cell death, as well as yet-unrelated homeostatic features, e.g. carbohydrate metabolism. A set of 14 genes linked STAT5Delta750 expression to the poorly differentiated carcinoma phenotype and STAT5ca to the highly differentiated papillary adenocarcinoma.Specifically affected genes exhibited differential expression in an individual tumor set vs. its counterpart and the intact mammary gland: 50 genes were specifically affected by STAT5ca, and 94% of these were downregulated, the latter involved in suppression of tumor suppressors and proliferation antagonistics. This substantial downregulation distinguishes the STAT5ca-induced tumorigenic consequences from the relatively equal effect of the STAT5Delta750 on gene expression, which included significant elevation in the expression of oncogenes and growth mediators.STAT5Delta750 mRNA expression was below detection levels in the tumors and the amount of STAT5ca transcript was not correlated with the expression of its specifically affected genes. Interestingly, we identified several groups of three to eight genes affected by a particular STAT5 variant with significant correlated expression at distinct locations in the clustergram.ConclusionThe different gene-expression profiles in mammary tumors caused by the STAT5Delta750 and STAT5ca variants, corroborated by the absence of a direct link to transgenic STAT5 expression, imply distinct metabolic consequences for their oncogenic role which probably initiate early in tumor development. Tumorigenesis may involve induction of growth factor and oncogenes by STAT5Delta750 or suppression of tumor suppressors and growth antagonists by STAT5ca. The list of genes specifically affected by the STAT5 variants may provide a basis for the development of a marker set for their distinct oncogenic role.

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Distinct expression and ligand-binding profiles of two constitutively active GPR17 splice variants.

Publications

Distinct expression and ligand-binding profiles of two constitutively active GPR17 splice variants.

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