Na(+)/Ca(2+) exchanger isoforms in nervous tissue and isoform-specific regulation by PKA
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Na+/Ca2+ exchanger isoforms in nervous tissue and isoform-specific regulation by PKAAbstract
In many tissues including nervous tissue, cytoplasmic Ca2+ concentration is regulated by the plasma membrane Na+/Ca{2+ exchanger. I have measured Na+/Ca2+ exchanger activity in brain-derived primary astrocyte cultures by using Na+-dependent45Ca2+ influx. Multiple Na+/Ca2+ exchanger genes have been identified in the rat brain tissue. I used RT-PCR cloning and RNase protection to show that NCX1 mRNA was the main transcript expressed in primary astrocyte and neuron cultures compared to NCX2. The NCX1 gene has previously been demonstrated to undergo alternative splicing of the primary transcript in the carboxyl end of the intracellular loop of the Na+/Ca2+ exchanger protein to produce multiple tissue-specific isoforms. This alternative splicing is generated by six exons present in the gene (A, B, C, D, E and F), among which exons A and B are mutually exclusive in their expression and the other four are "cassette" type exons. Using RT-PCR cloning, I demonstrated the presence of four NCX1 isoforms (BDEF, BDF, BDE and BD) in the cultured astrocytes and two NCX1 isoforms (ADF and AD) in the cultured neurons. To quantitate the relative mRNA amounts of these NCX1 isoforms, I developed a new method, quantitative end-labeled RT-PCR (QERT-PCR). Using this method, I have demonstrated that three exon B-containing isoforms (BDEF, BDF and BD) are predominant in the primary astrocyte culture and each represents about 23% of the total QERT-PCR product. Interestingly, QERT-PCR analysis of the C6 astrocytoma cell line has shown a similar pattern for the predominant isoforms as in the primary astrocyte culture. In the primary neuron culture, QERT-PCR results demonstrate that two exon A-containing isoforms (ADF and AD) are the predominant representing 57% and 37% of the NCX1 transcripts, respectively. While alternative splicing of NCX1 would predict that up to 32 different Na+/Ca2+ exchanger mRNAs may be produced by various combination of exons A to F, by using QERT-PCR, I have demonstrated there are a total of only 16 isoforms including the 3 predominant ones in the primary astrocyte culture. Each of these isoforms contain either exon A or B and all have exon D in the sequence. To determine the functional significance underlying the different isoforms in these cells, a representative astrocytic isoform (BD) and neuronal isoform (AD) have been subcloned into the full-length NCX1 cDNA. When RNA from these constructs were expressed in Xenopus oocytes, both isoforms showed the Na+/Ca2+ exchanger activity. Importantly, the activity of the AD isoform increased by 31-54% after activating the protein kinase A (PKA) pathway whereas the activity of the BD isoform did not change. Function of chimeric constructs containing parts of exon A and B sequences have been examined, demonstrating that the carboxyl terminal 20 amino acids of exon A were important to enable PKA to increase the Na+/Ca2+ exchanger activity. Additionally, experiments with exon D deletion construct showed that this exon was also necessary for the PKA-dependent modulation of the Na+/Ca2+ exchanger activity.Description
University of Maryland, Baltimore. Microbiology and Immunology. Ph.D. 1998Keyword
Biology, Molecularisoform-specific regulation
PKA
Cyclic AMP-Dependent Protein Kinases
Nerve Tissue
Sodium-Calcium Exchanger--genetics
Identifier to cite or link to this item
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Functional Characterization of CXCR3 Receptor Isoforms in Breast CancerLi, Yanchun; Fulton, Amy M. (2012)The chemokine receptor CXCR3 belongs to the G protein-coupled receptor family. Chemokine receptors direct the trafficking of hematopoietic cells, but several are expressed on malignant cells where they play a role in tumor growth and dissemination. CXCR3 binds to the ligands CXCL9, 10, 11 and CXCL4. CXCR3 is up-regulated in breast cancers and is associated with poor prognosis, and inhibiting CXCR3 can reduce lung metastasis in a preclinical model of breast cancer. The detailed molecular mechanisms by which CXCR3 activation promotes breast cancer invasiveness remain to be clarified. In addition, the existence of two splice isoforms of CXCR3 (CXCR3-A and CXCR3-B) has made the picture more complex. In renal cell cancer, CXCR3-A has pro-neoplastic properties such as promoting proliferation and migration, whereas CXCR3-B inhibits proliferation and exerts no chemotactic effect. CXCR3-A and CXCR3-B are expressed in both normal and malignant mammary epithelial cells; CXCR3-B expression was reduced in breast cancer cell lines compared to benign cells. We hypothesize that the expression pattern shift between the two isoforms contributes to the malignant behavior of breast cancer. The purpose of the current study was to characterize the functions of CXCR3 in breast cancer cells and to determine how the shift in balance between the two isoforms affects the invasiveness of breast cancer cells. We have overexpressed and silenced expression of CXCR3-B in the highly metastatic human breast cancer cell line MDA-MB-231. Ectopic CXCR3-B expression resulted in decreased ligand-induced ERK1/2 and p38 phosphorylation, compared to enhancement of phosphorylation of the two pathways in parental MDA-MB-231 cells. Interestingly, suppression of CXCR3-B gene expression did not change the signaling response. Likewise, migratory and invasive abilities of MDA-MB-231 cells were attenuated by CXCR3-B over-expression, but not affected by CXCR3-B gene-silencing. These data suggested CXCR3-B may function to antagonize the contribution of CXCR3-A to invasiveness of cancer cells. We also identified pro-tumor activities of CXCR3-B. CXCR3-B gene expression was highly up-regulated in breast cancer stem-like cells, and overexpression of CXCR3-B supported mammosphere formation. Taken together, these data support a complex role for CXCR3-B; inhibiting the migration and invasion role of CXCR3-A but also supporting a minority population with tumor-initiating potential.
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