Subunit oligomerization, and topology of the inositol 1,4, 5-trisphosphate receptor.

The inositol 1,4,5-trisphosphate receptor (InsP(3)R) is a tetrameric assembly of highly conserved subunits that contain multiple membrane-spanning sequences in the C-terminal region of the protein. In studies aimed at investigating the oligomerization and transmembrane topology of the type-1 InsP(3)R, a series of membrane-spanning region truncation and deletion plasmids were constructed. These plasmids were transiently transfected in COS-1 cells, and the resulting expression products were analyzed for the ability to assemble into tetrameric structures. The topology of the membrane-spanning region truncations and the full-length receptor was determined by immunocytochemical analysis of transfected COS-1 cells using complete or selective permeabilization strategies. Our results are the first to experimentally define the presence of six membrane-spanning regions. These results are consistent with the current model for the organization of the InsP(3)R in the endoplasmic reticulum and show that the truncation mutants are properly targeted and oriented in the endoplasmic reticulum membrane, thus making them amenable reagents to study receptor subunit oligomerization. Fractionation of soluble and membrane protein components revealed that the first two membrane-spanning regions were necessary for membrane targeting of the receptor. Sedimentation and immunoprecipitation experiments show that assembly of the receptor subunits was an additive process as the number of membrane-spanning regions increased. Immunoprecipitations from cells co-expressing the full-length receptor and carboxyl-terminal truncations reveal that constructs expressing the first two or more membrane-spanning domains were capable of co-assembling with the full-length receptor. Inclusion of the fifth membrane-spanning segment significantly enhanced the degree of oligomerization. Furthermore, a deletion construct containing only membrane-spanning regions 5 and 6 oligomerized to a similar extent as that of the wild type protein. Membrane-spanning region deletion constructions that terminate with the receptor's 145 carboxyl-terminal amino acids were found to have enhanced assembly characteristics and implicate the carboxyl terminus as a determinant in oligomerization. Our results reveal a process of receptor assembly involving several distinct yet additive components and define the fifth and sixth membrane spanning regions as the key determinants in receptor oligomerization.

Simulated microgravity conditions enhance differentiation of cultured PC12 cells towards the neuroendocrine phenotype.

We are studying microenvironmental cues which contribute to neuroendocrine organ assembly and tissue-specific differentiation. As our in vitro model, we cultured rat adrenal medullary PC12 pheochromocytoma cells in a novel cell culture system, the NASA rotating wall vessel (RWV) bioreactors. This "simulated microgravity" environment in RWV bioreactors, characterized by randomizing gravitational vectors and minimizing shear stress, has been shown to favor macroscopic tissue assembly and to induce tissue-specific differentiation. We hypothesized that the unique culture conditions in the RWV bioreactors might enhance the in vitro formation of neuroendocrine organoids. To test our hypothesis, we evaluated the expression of several markers of neuroendocrine differentiation in cultures of PC12 cells maintained for up to 20 d in the slow turning lateral vessel (STLV) type RWV. PC12 cell differentiation was assessed by morphological, immunological, biochemical and molecular techniques. PC12 cells, cultured under "simulated microgravity" conditions, formed macroscopic, tissue-like organoids several millimeters in diameter. Concomitantly, the expression of phenylethanolamine-N-methyl transferase (PNMT), but not of other catecholamine synthesizing enzymes, was enhanced. Increased PNMT expression, as verified on both the gene and protein level, was accompanied by an increase in the specific activity of the enzyme. Furthermore, after 20 d in culture in the STLV, we observed altered patterns of protein tyrosine phosphorylation and prolonged activation of c-fos, a member of the AP-1 nuclear transcription factor complex. We conclude that culture conditions in the RWV appear to selectively activate signal transduction pathways leading to enhanced neuroendocrine differentiation of PC12 cells.

Membrane potential mediates H(+)-ATPase dependence of "degradative pathway" endosomal fusion.

In some epithelial cell lines, the uptake and degradation of proteins is so pronounced as to be regarded as a specialized function known as "degradative endocytosis." The endosomal pathways of the renal proximal tubule and the visceral yolk sac share highly specialized structures for "degradative endocytosis." These endosomal pathways also have a unique distribution of their H(+)-ATPase, predominantly in the subapical endosomal pathway. Previous studies provide only indirect evidence that H(+)-ATPases participate in endosomal fusion events: formation of vesicular intermediates between early and late endosomes is H(+)-ATPase dependent in baby hamster kidney cells, and H(+)-ATPase subunits bind fusion complex proteins in detergent extracts of fresh rat brain. To determine directly whether homotypic endosomal fusion is H(+)-ATPase dependent, we inhibited v-type H(+)-ATPase during flow cytometry and cuvette-based fusion assays reconstituting endosomal fusion in vitro. We report that homotypic fusion in subapical endosomes derived from rat renal cortex, and immortalized visceral yolk sac cells in culture, is inhibited by the v-type H(+)-ATPase specific inhibitor bafilomycin A1. Inhibition of fusion by H(+)-ATPase is mediated by the membrane potential as collapsing the pH gradient with nigericin had no effect on homotypic endosomal fusion, while collapsing the membrane potential with valinomycin inhibited endosomal fusion. Utilizing an in vitro reconstitution assay this data provides the first direct evidence for a role of v-type H(+)-ATPase in mammalian homotypic endosomal fusion.