Preserving Creativity in Medicine

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Regulation of the G-protein αi-2 subunit gene in LLC-PK1 renal cells and isolation of porcine genomic clones encoding the gene promoter

Heterotrimeric G-proteins function as signal transducers for a variety of hormone-coupled enzyme and ion transport systems in eukaryotic cells. We have studied G-protein-coupled processes that appear to be developmentally regulated in polarized pig kidney cells (LLC-PK1). Following trypsinization, LLC-PK1 cells differentiate from a rounded cell type to a fully polarized epithelium by 7 days of culture. During this differentiation, the expression of G-protein alpha-i-2 subunit mRNA was not detected until day 4 of culture, it peaked at day 6, and declined thereafter. In contrast, G-protein alpha-(s) subunit mRNA which peaked on day 4 was easily detected on all culture days. The presence of the alpha-i-2 protein on epithelial cell basolateral membranes followed the same pattern of mRNA expression during culture. To understand the developmental expression of the alpha-i-2 subunit in non-polarized cells and its potential regulation by hormones and second messengers in polarized cells at the transcriptional level, genomic DNA segments encoding the alpha-i-2 gene promoter were isolated from an EMBL-3 porcine genomic library. S1 nuclease analysis of LLC-PK1 mRNA with cRNA probes derived from these DNA segments revealed major and a minor transcriptional start sites 131 and 171 base pairs upstream of the translation initiation site. The porcine and human alpha-i-2 subunit genes shared a 78% sequence identity in their 5' flanks which suggested an evolutionary conversation of cis elements required to influence their transcription. The porcine alpha-i-2 gene promoter was identified by fusing DNA segments encoding putative 5'-flanking areas of the gene to a plasmid that contained a firefly luciferase reporter gene but lacked a promoter. The minimal promoter was found between -130 and -60 base pairs from the major transcription start site. No typical "TATA-like" sequences were found. However, a "GC" box and a "TGTGG" sequence were two potential cis elements required for basal transcription of the porcine gene promoter which shared a 76% sequence identity to the promoter of another GTP-binding protein, the human c-Ha-ras proto-oncogene. Transcription of the gene was inhibited following treatment of renal cells with 10(-8) M dexamethasone. These studies suggest that alpha-i-2 gene expression is regulated in LLC-PK1 cells. Identification of the gene's promoter and 5'-flanking sequences provide a basis for elucidating "cis-acting" DNA sequences and "trans-acting" protein factors that act in concert to control the transcriptional regulation of the alpha-i-2 gene which may modulate G-protein coupled effector responses in vasopressin-sensitive renal epithelia

Structural model of a2-subunit N-terminus and its binding interface for Arf-GEF CTH2: Implication for regulation of V-ATPase, CTH2 function and rational drug design

We have previously identified the interaction between mammalian V-ATPase a2-subunit isoform and cytohesin-2 (CTH2) and studied molecular details of binding between these proteins. In particular, we found that six peptides derived from the N-terminal cytosolic domain of a2 subunit (a2N1–402) are involved in interaction with CTH2 (Merkulova, Bakulina, Thaker, Grüber, & Marshansky, 2010). However, the actual 3D binding interface was not determined in that study due to the lack of high-resolution structural information about a-subunits of V-ATPase. Here, using a combination of homology modeling and NMR analysis, we generated the structural model of complete a2N1–402 and uncovered the CTH2-binding interface. First, using the crystal-structure of the bacterial M. rubber Icyt-subunit of A-ATPase as a template (Srinivasan, Vyas, Baker, & Quiocho, 2011), we built a homology model of mammalian a2N1–352 fragment. Next, we combined it with the determined NMR structures of peptides a2N368–395 and a2N386–402 of the C-terminal section of a2N1–402. The complete molecular model of a2N1–402 revealed that six CTH2 interacting peptides are clustered in the distal and proximal lobe sub-domains of a2N1–402. Our data indicate that the proximal lobe sub-domain is the major interacting site with the Sec7 domain of first CTH2 protein, while the distal lobe sub-domain of a2N1–402 interacts with the PH-domain of second CTH2. Indeed, using Sec7/Arf-GEF activity assay we experimentally confirmed our model. The interface formed by peptides a2N1–17 and a2N35–49 is involved in specific interaction with Sec7 domain and regulation of GEF activity. These data are critical for understanding of the cross-talk between V-ATPase and CTH2 as well as for the rational drug design to regulate their function

Purified epithelial Na+ channel complex contains the pertussis toxin-sensitive Gαi-3 protein

We have recently demonstrated that the amiloride-sensitive Na+ channel in the apical membrane of the renal epithelial cell line, A6, is modulated by the alpha(i-3) subunit of the G(i-3) protein. We also showed that a 700-kDa protein complex can be purified from the membranes of A6 epithelia which (a) can reconstitute the amiloride-sensitive Na+ influx in liposomes and planar bilayer membranes and (b) consists of six major protein bands observed on reducing sodium dodecyl sulfate-polyacrylamide gels with molecular masses ranging from 35 to 320 kDa. The present study was undertaken to determine if the alpha(i-3) subunit was a member of this Na+ channel complex. G-alpha(i) structure and function were identified by Western blotting with specific G-alpha(i) subunit antibodies and Na+ channel antibodies, through ADP-ribosylation with pertussis toxin, and by immunocytochemical localization of the Na+ channel and G-alpha(i) proteins. We demonstrate that two protein substrates are ADP-ribosylated in the 700-kDa complex in the presence of pertussis toxin and are specifically immunoprecipitated with an anti-Na+ channel polyclonal antibody. One of these substrates, a 41-kDa protein, was identified as the alpha(i-3) subunit of the G(i-3) protein on Western blots with specific antibodies. Na+ channel antibodies do not recognize G-alpha(i-3) on Western blots of Golgi membranes which contain alpha(i-3) but not Na+ channel proteins, nor do they immunoprecipitate alpha(i-3) from solubilized Golgi membranes; however, alpha(i-3) is coprecipitated as part of the Na+ channel complex from A6 cell membranes by polyclonal Na+ channel antibodies. Both alpha(i-3) and the Na+ channel have been localized in A6 cells by confocal imaging and immunofluorescence with specific antibodies and are found to be in distinct but adjacent domains of the apical cell surface. In functional studies, alpha(i-3), but not alpha(i-2), stimulates Na+ channel activity. These data are therefore consistent with the localization of Na+ channel activity and modulatory alpha(i-3) protein at the apical plasma membrane, which together represent a specific signal transduction pathway for ion channel regulation

Vasopressin receptor-mediated functional signaling pathway in primary cilia of renal epithelial cells

The primary cilium of renal epithelial cells is a nonmotile sensory organelle, implicated in mechanosensory transduction signals. Recent studies from our laboratory indicate that renal epithelial primary cilia display abundant channel activity; however, the presence and functional role of specific membrane receptors in this organelle are heretofore unknown. Here, we determined a functional signaling pathway associated with the type 2 vasopressin receptor (V2R) in primary cilia of renal epithelial cells. Besides their normal localization on basolateral membrane, V2R was expressed in primary cilia of LLC-PK1 renal epithelial cells. The presence of V2R in primary cilia was determined by spontaneous fluorescence of a V2R-gfp chimera and confirmed by immunocytochemical analysis of wild-type LLC-PK1 cells stained with anti-V2R antibodies and in LLC-PK1 cells overexpressing the V2R-Flag, with anti-Flag antibody. Ciliary V2R colocalized with adenylyl cyclase (AC) type V/VI in all cell types tested. Functional coupling of the receptors with AC was confirmed by measurement of cAMP production in isolated cilia and by testing AVP-induced cation-selective channel activity either in reconstituted lipid bilayers or subjected to membrane-attached patch clamping. Addition of either 10 μM AVP (trans) or forskolin (cis) in the presence but not the absence of ATP (1 mM, cis) stimulated cation-selective channel activity in ciliary membranes. This channel activity was reduced by addition of the PKA inhibitor PKI. The data provide the first demonstration for the presence of V2R in primary cilia of renal epithelial cells, and a functional cAMP-signaling pathway, which targets ciliary channel function and may help control the sensory function of the primary cilium.Fil: Raychowdhury, Malay K.. Massachusetts General Hospital; Estados Unidos. Harvard Medical School; Estados UnidosFil: Ramos, Arnolt J.. Massachusetts General Hospital; Estados Unidos. Harvard Medical School; Estados UnidosFil: Zhang, Peng. Massachusetts General Hospital; Estados Unidos. Harvard Medical School; Estados UnidosFil: McLaughin, Margaret. Harvard Medical School; Estados Unidos. Massachusetts General Hospital; Estados UnidosFil: Dai, Xiao-Qing. University of Alberta; CanadáFil: Chen, Xing-Zhen. University of Alberta; CanadáFil: Montalbetti, Nicolas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Cardiológicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Cardiológicas; ArgentinaFil: Cantero, Maria del Rocio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Cardiológicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Cardiológicas; ArgentinaFil: Ausiello, Dennis A.. Massachusetts General Hospital; Estados Unidos. Harvard Medical School; Estados UnidosFil: Cantiello, Horacio Fabio. Massachusetts General Hospital; Estados Unidos. Harvard Medical School; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Cardiológicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Cardiológicas; Argentin