Absence of aquaporin-4 water channels from kidneys of the desert rodent Dipodomys merriami merriami.

Recently, we found that aquaporin-4 (AQP4) is expressed in the S3 segment of renal proximal tubules of mice but not in rat proximal tubules. Because mice have relatively larger papillae than rats, it was proposed that the renal distribution of AQP4 in various species could be related to their maximum urinary concentrating ability. Therefore, kidneys and other tissues of Merriam's desert kangaroo rat, Dipodomys merriami merriami, which produce extremely concentrated urine (up to 5,000 mosmol/kgH(2)O), were examined for AQP4 expression and localization. Contrary to our expectation, AQP4 immunostaining was undetectable in any region of the kidney, and the absence of AQP4 protein was confirmed by Western blotting. By freeze fracture electron microscopy, orthogonal arrays of intramembraneous particles (OAPs) were not detectable in plasma membranes of principal cells and proximal tubules. However, AQP4 protein was readily detectable in gastric parietal and brain astroglial cells. Northern blotting failed to detect AQP4 mRNA in kangaroo rat kidneys, whereas both in situ hybridization and RT-PCR experiments did reveal AQP4 mRNA in collecting ducts and proximal tubules of the S3 segment. These results suggest that renal expression of AQP4 in the kangaroo rat kidney is regulated at the transcriptional or translational level, and the absence of AQP4 may be critical for the extreme urinary concentration that occurs in this species.

Gene 33/Mig-6, a transcriptionally inducible adapter protein that binds GTP-Cdc42 and activates SAPK/JNK. A potential marker transcript for chronic pathologic conditions, such as diabetic nephropathy. Possible role in the response to persistent stress.

Chronic stresses, including the mechanical strain caused by hypertension or excess pulmonary ventilation pressure, lead to important clinical consequences, including hypertrophy and acute respiratory distress syndrome. Pathologic hypertrophy contributes to decreased organ function and, ultimately, organ failure; and cardiac and diabetic renal hypertrophy are major causes of morbidity and morality in the developed world. Likewise, acute respiratory distress syndrome is a serious potential side effect of mechanical pulmonary ventilation. Whereas the deleterious effects of chronic stress are well established, the molecular mechanisms by which these stresses affect cell function are still poorly characterized. gene 33 (also called mitogen-inducible gene-6, mig-6) is an immediate early gene that is transcriptionally induced by a divergent array of extracellular stimuli. The physiologic function of Gene 33 is unknown. Here we show that gene 33 mRNA levels increase sharply in response to a set of commonly occurring chronic stress stimuli: mechanical strain, vasoactive peptides, and diabetic nephropathy. Induction of gene 33 requires the stress-activated protein kinases (SAPKs)/c-Jun NH(2)-terminal kinases. This expression pattern suggests that gene 33 is a potential marker for diabetic nephropathy and other pathologic responses to persistent sublethal stress. The structure of Gene 33 indicates an adapter protein capable of binding monomeric GTPases of the Rho subfamily. Consistent with this, Gene 33 interacts in vivo and, in a GTP-dependent manner, in vitro with Cdc42Hs; and transient expression of Gene 33 results in the selective activation of the SAPKs. These results imply a reciprocal, positive feedback relationship between Gene 33 expression and SAPK activation. Expression of Gene 33 at sufficient levels may enable a compensatory reprogramming of cellular function in response to chronic stress, which may have pathophysiological consequences.

Molecular identification of I1PP2A, a novel potent heat-stable inhibitor protein of protein phosphatase 2A.

The amino acid sequences of two tryptic peptides derived from purified preparations of I1PP2A indicated that this potent heat-stable protein inhibitor of protein phosphatase 2A (PP2A) may be equivalent to putative histocompatibility leukocyte antigens class II-associated protein I (PHAP-I). Experiments using purified preparations of recombinant human PHAP-I confirmed that this protein inhibited PP2A. Half-maximal inhibition of the phosphatase occurred at about 4 nM PHAP-I, similar to the half-maximal inhibition obtained with purified preparations of bovine kidney I1PP2A. In addition, PHAP-I did not affect the activities of protein phosphatase 1, 2B, and 2C in a manner analogous to that of I1PP2A. Together, the results establish the identity of I1PP2A on a firm basis.

The myeloid leukemia-associated protein SET is a potent inhibitor of protein phosphatase 2A.

Two potent heat-stable protein phosphatase 2A (PP2A) inhibitor proteins designated I1PP2A and I2PP2A have been purified to apparent homogeneity from extracts of bovine kidney (Li, M., Guo, H., and Damuni, Z. (1995) Biochemistry 34, 1988-1996). N-terminal and internal amino acid sequencing indicated that I2PP2A was a truncated form of SET, a largely nuclear protein that is fused to nucleoporin Nup214 in acute non-lymphocytic myeloid leukemia. Experiments using purified preparations of recombinant human SET confirmed that this protein inhibited PP2A. Half-maximal inhibition of the phosphatase occurred at about 2 nM SET. By contrast, SET (up to 20 nM) did not affect the activities of purified preparations of protein phosphatases 1, 2B, and 2C. The results indicate that SET is a potent and specific inhibitor of PP2A and suggest that impaired regulation of PP2A may contribute to acute myeloid leukemogenesis.

Phosphorylation of eukaryotic protein synthesis initiation factor 4E by insulin-stimulated protamine kinase.

Insulin-stimulated protamine kinase (cPK) and protein kinase C (PKC) phosphorylated eukaryotic protein synthesis initiation factor 4E (eIF-4E) on serine and threonine residues located on an identical tryptic fragment as judged by two-dimensional phosphopeptide mapping. With cPK and PKC, the apparent Km for eIF-4E was about 1.2 and 50 microM, respectively. Relative to recombinant human eIF-4E, cPK exhibited about 100% and < or = 5% activity with eIF-4ES209A and eIF-4ET210A, respectively, and eIF-4ES209A was phosphorylated exclusively on threonines. Bovine kidney eIF-4E enhanced up to 1.8-fold globin synthesis in m7GTP-Sepharose-treated reticulocyte lysates. In contrast, following incubation with cPK, these eIF-4E preparations stimulated globin synthesis up to 6-fold. Compared to the dephosphorylation of the cPK-modified serine on eIF-4E, reticulocyte lysates and highly purified protein phosphatase 2A exhibited marked preference for the cPK-modified threonine. The results indicate that cPK phosphorylates eIF-4E on Ser209 and Thr210, that the hydroxyl group or phosphorylation of Thr210 is necessary for cPK to act on Ser209, and that Ser209 phosphorylation activates reticulocyte globin synthesis. The results suggest that cPK could contribute to the insulin-stimulated phosphorylation of eIF-4E, but that protein phosphatase 2A may confer the site specificity of this response.

The beta 4 subunit of the integrin family is displayed on a restricted subset of endothelium in mice.

More than 15 subunits of the integrin family of cell surface adhesion molecules have been identified. The alpha 6 beta 4 integrin has recently been identified as a component of hemidesmosomes of stratified squamous epithelium. The monoclonal antibody (mAb) 346-11A binds to the beta 4 subunit in mice. Sequence analysis of a cDNA clone coding for this epitope localizes reaction of the mAb to a portion about half way through the extracellular domain at the beginning of the cysteine-rich region. Sites of beta 4 expression in mice were detected by autoradiographic analysis of tissues collected from mice 24 or 48 h after intravenous injection of 125I-labeled mAb 346-11A. This non-quantitative technique emphasizes detection of antigen exposed in the vascular space. These data show that in addition to the epithelia of several organs, the endothelia of intermediate vessels throughout the body are sites of beta 4 expression. In particular, endothelium of larger vessels but not capillaries in lung, vessels in thymus, spleen and Peyer's patches, and portal vessels but not the central veins of the liver, are positive. The expression of the beta 4 integrin in blood vessels may indicate a specialized function for beta 4 at these sites that is distinct from its role in hemidesmosome-mediated attachment.

Molecular cloning and analysis of full-length cDNAs cognate to a rat gene under multihormonal control.

Gene 33 is a multihormonally regulated rat gene whose transcription is rapidly and markedly enhanced by glucocorticoids, insulin, or cAMP. A cDNA clone (p216) containing a nearly full-length DNA complementary to the mRNA of this gene was isolated from a cDNA library and sequenced. The cDNA represents the entire mRNA transcript, except for three bases at the 5' terminus. The message is 2970 nucleotides long and can encode a protein of 459 amino acids with a molecular mass of 49,919 Da. The deduced amino acid sequence is rich in proline and serine but bears little homology to any known sequences. The 5' untranslated region, rich in G + C, is 270 nucleotides long and contains two apparently nonfunctional AUG initiator codons. The 3' untranslated sequence, rich in A + U, is 1323 nucleotides long and has a polyadenylation signal 13 bases upstream of the poly(A) tract. Two mRNA transcripts of the gene, which appear to be the consequence of alternative usage of splice sites, have been identified. The less abundant mRNA is truncated by 228 nucleotides in the protein coding region. The reading frame is maintained and this mRNA can code for a protein of 383 amino acids with a calculated molecular mass of 42,204 Da.