GenBank NM Research Articles

Molecular cloning and characterization of mouse aquaporin 6

In the rat kidney, aquaporin (AQP) 6 is localized in the intracellular vesicle membranes of type-A intercalated cells of the collecting duct; mouse AQP6 (mAQP6) has not been characterized. Although mAQP6 was originally cloned from cDNA in a mouse cerebellum library (GenBank NM 175087), we have independently cloned a cDNA encoding mAQP6 from an adult kidney cDNA library (C57BL/6J strain). We identified two different spliced variants of mAQP6: mAQP6a and mAQP6b. The mAQP6a isoform is almost identical to that of rat AQP6, whereas mAQP6b is identical to that reported in the mouse cerebellum library mentioned above. We found that the mRNA expression of these two spliced variants is regulated in a tissue-specific and age-dependent manner. Functional analyses of water and ion permeation revealed that mAQP6a functions like rat AQP6 and that mAQP6b does not function as either a water channel or an ion channel under our experimental conditions.

Hemoglobin Is Expressed by Alveolar Epithelial Cells

Hemoglobin gene expression in non-erythroid cells has been previously reported in activated macrophages from adult mice and lens cells, and recent studies indicate that alveolar epithelial cells can be derived from hematopoietic stem cells. Our laboratory has now produced strong evidence that hemoglobin is expressed by alveolar type II (ATII) cells and Clara cells, the primary producers of pulmonary surfactant. ATII cells are also closely involved in innate immunity within the lung and are stem cells that differentiate into alveolar type I cells. Reverse transcriptase-PCR was used to measure the expression of transcripts from the alpha- and beta-globin gene clusters in several human and rodent pulmonary epithelial cells. Surprisingly, the two major globin mRNAs characteristic of adult erythroid precursor cells were clearly expressed in human A549 and H441 cell lines, mouse MLE-15 cells, and primary ATII cells isolated from normal rat and mouse lungs. DNA sequencing verified that these PCR products were indeed the result of specific amplification of globin gene cDNAs. These alveolar epithelial cells also expressed the corresponding hemoglobin protein subunits as determined by Western blotting, and tandem mass spectrometry sequencing was used to verify the presence of both alpha- and beta-globin polypeptides in rat primary ATII cells. The function of hemoglobin expression by cells of the pulmonary epithelium will be determined by future studies, but this novel finding could potentially have important implications for the physiology and pathology of the lung.

Identification of a novel alternative splicing variant of RGS5 mRNA in human ocular tissues

Regulator of G protein signaling (RGS) proteins act as GTPase-activating proteins (GAPs) for Galpha subunits and negatively regulate G protein-coupled receptor signaling. Using RGS5 gene-specific RT-PCR, we have identified a novel alternative splicing variant of RGS5 mRNA in human ocular tissues. The alternative splicing of RGS5 mRNA occurred at position +44 (GenBank NM_003617), spliced out 174 bp (+44 to +218 bp) of the coding region, and encoded an RGS5s protein with a 108 amino acid N-terminal deletion. This study is the first to document alternative splicing of an RGS5 gene. We therefore studied RGS5 and RGS5s mRNA distribution in human tissues. In the eye, RGS5s was found to be highly expressed in the ciliary body and trabecular meshwork. It was also expressed in the kidney, brain, spleen, skeletal muscle and small intestine, but was not detectable in the liver, lung, heart. RGS5s was not found in monkey and rat ocular tissues, indicating species specificity for the eye. Comparing the recombinant RGS5 and RGS5s expression in HEK293/EBNA cells, RGS5s was present almost exclusively in the cytosolic fraction, whereas RGS5 was present in both membrane and cytosolic fractions. The data suggest that the N-terminal of RGS5 may be important for protein translocation to the cell membrane. Both RGS5 and RGS5s antagonized the rapid phosphorylation of p44/42 MAP kinase induced by Galphai coupled cannibinoid receptor-1 activation. RGS5, but not RGS5s, inhibited the Ca2+ signaling initiated by activation of Galphaq coupled angiotensin II receptors (AT1) and prostaglandin FP receptors. Cotransfection of RGS5s with RGS5 resulted in the blockade of RGS5 actions with respect to inhibition of the signal transduction initiated by activation of both AT1 and FP receptor, suggesting that RGS5s may contain functional domains that compete with RGS5 in the regulation of the Galphaq coupled AT1 and FP receptors. The unique expression pattern, cellular localization and functions of RGS5s suggest that RGS5s may play a critical role in the regulation of intracellular signaling pathways.

A genetic polymorphism in the coding region of the gastric intrinsic factor gene (GIF) is associated with congenital intrinsic factor deficiency.

Congenital intrinsic factor (IF) deficiency is a disorder characterized by megaloblastic anemia due to the absence of gastric IF (GIF, GenBank NM_005142) and GIF antibodies, with probable autosomal recessive inheritance. Most of the reported patients are isolated cases without genetic studies of the parents or siblings. Complete exonic sequences were determined from the PCR products generated from genomic DNA of five affected individuals. All probands had the identical variant (g.68A>G) in the second position of the fifth codon in the coding sequence of the gene that introduces a restriction enzyme site for Msp I and predicts a change in the mature protein from glutamine(5) (CAG) to arginine(5) (CGG). Three subjects were homozygous for this base exchange and two subjects were heterozygous, one of which was apparently a compound heterozygote at positions 1 and 2 of the fifth codon ([g.67C>G] + [g.68A>G]). The other patient, heterozygous for position 2, had one heterozygous unaffected parent. Most parents were heterozygous for this base exchange, confirming the pattern of autosomal recessive inheritance for congenital IF deficiency. cDNA encoding GIF was mutated at base pair g.68 (A>G) and expressed in COS-7 cells. The apparent size, secretion rate, and sensitivity to pepsin hydrolysis of the expressed IF were similar to native IF. The allelic frequency of g.68A>G was 0.067 and 0.038 in two control populations. This sequence aberration is not the cause of the phenotype, but is associated with the genotype of congenital IF deficiency and could serve as a marker for inheritance of this disorder.