Biomedical subjects

Herbert Spring

Publications and source records attributed to Herbert Spring.

22 records · Page 2Linked to original sources

PubMed2002

Structural requirements for the apical sorting of human multidrug resistance protein 2 (ABCC2).

The human multidrug resistance protein 2 (MRP2, symbol ABCC2) is a polytopic membrane glycoprotein of 1545 amino acids which exports anionic conjugates across the apical membrane of polarized cells. A chimeric protein composed of C-proximal MRP2 and N-proximal MRP1 localized to the apical membrane of polarized Madin-Darby canine kidney cells (MDCKII) indicating involvement of the carboxy-proximal part of human MRP2 in apical sorting. When compared to other MRP family members, MRP2 has a seven-amino-acid extension at its C-terminus with the last three amino acids (TKF) comprising a PDZ-interacting motif. In order to analyze whether this extension is required for apical sorting of MRP2, we generated MRP2 constructs mutated and stepwise truncated at their C-termini. These constructs were fused via their N-termini to green fluorescent protein (GFP) and were transiently transfected into polarized, liver-derived human HepG2 cells. Quantitative analysis showed that full-length GFP-MRP2 was localized to the apical membrane in 73% of transfected, polarized cells, whereas it remained on intracellular membranes in 27% of cells. Removal of the C-terminal TKF peptide and stepwise deletion of up to 11 amino acids did not change this predominant apical distribution. However, apical localization was largely impaired when GFP-MRP2 was C-terminally truncated by 15 or more amino acids. Thus, neither the PDZ-interacting TKF motif nor the full seven-amino-acid extension were necessary for apical sorting of MRP2. Instead, our data indicate that a deletion of at least 15 C-terminal amino acids impairs the localization of MRP2 to the apical membrane of polarized cells.

Amino Acid Sequence↗

PubMed2002

Immunolocalization of multidrug resistance protein 5 in the human genitourinary system.

PURPOSE: The intracellular messenger cyclic guanosine monophosphate (cGMP) has an important role in regulating smooth muscle tone. An increase in intracellular cGMP levels is a prerequisite for penile erection. Inhibition of cGMP degradation by cGMP specific phosphodiesterase 5 has been used for treating erectile dysfunction. In addition to degradation by phosphodiesterase, cGMP is exported from cells by multidrug resistance protein 5 (MRP5), also called ABCC5, which we recently identified as an adenosine triphosphate dependent export pump for cGMP. MRP5 is potently inhibited by substances known as phosphodiesterase inhibitors, including sildenafil and trequinsin. Therefore, we analyzed whether MRP5 is expressed in tissues of the human genitourinary system and whether MRP5 and phosphodiesterase 5 proteins are localized in the same cell types. MATERIALS AND METHODS: Localization of MRP5 and phosphodiesterase 5 was analyzed by immunofluorescence microscopy in cryosections of various tissues of the human genitourinary system. RESULTS: MRP5 and phosphodiesterase 5 were co-expressed in smooth muscle cells of the corpus cavernosum, ureter, urethra and bladder. In addition, MRP5 and phosphodiesterase 5 were localized in epithelial cells of the mucosa in the ureter and urethra, and in blood vessels of the lamina propria. CONCLUSIONS: The co-expression of MRP5 and phosphodiesterase 5 in smooth muscle cells of the genitourinary system indicates 2 distinct pathways for cGMP removal. Thus, MRP5 inhibition represents a new approach for enhancing cGMP levels in smooth muscle cells and developing drugs for erectile dysfunction.

Adult↗

PubMed2001

Fully functional, naturally occurring and C-terminally truncated variant human immunodeficiency virus (HIV) Vif does not bind to HIV Gag but influences intermediate filament structure.

A variant human immunodeficiency virus type 1 (HIV-1) vif gene, vifA45-2, which encodes a protein lacking 19 amino acids at the C terminus but which is fully functional in supporting HIV replication in non-permissive cells has been described previously. By employing newly generated anti-VifA45 serum, further properties of VifA45 and its full-length counterpart, VifA45open, in comparison to Vif from HIV strain BH10 are reported in permissive HeLa and COS-7 cells. The results obtained using confocal microscopic localization studies and in vitro binding assays do not support a requirement for the direct interaction of HIV Gag with Vif. Furthermore and in contrast to previous conclusions, detergent solubility analyses do not demonstrate a role for the C terminus of Vif in mediating localization to the fraction containing cellular membrane proteins. Localization of Vif from HIV strain BH10 to perinuclear aggregates in a small fraction (about 10%) of transfected HeLa cells has been previously reported. The intermediate filament protein vimentin colocalizes to these structures. In contrast, VifA45 and VifA45open form perinuclear aggregates in nearly all transfected HeLa cells; vimentin as well as the cytoskeletal-bridging protein plectin, but not the microtubular protein tubulin, become relocalized to these structures. Interestingly, in COS-7 cells, all of the functional Vif proteins tested (Vif from strain BH10, VifA45 and VifA45open) predominantly localize in the cytoplasm but still induce dramatic aggregation of vimentin and plectin, i.e. in these cells the respective Vif proteins are influencing intermediate filament structure in the absence of colocalization.

Animals↗

PubMedDate not supplied

Investigation of nuclear architecture with a domain-presenting expression system.

We have investigated the topogenic properties of the nucleus by ectopic expression of chimeric proteins consisting of a NLS-modified cytoplasmic filament-forming protein, Xenopus laevis vimentin, and domains of inner nuclear membrane proteins. Whereas the "carrier" without cargo, the NLS-vimentin alone, is deposited in a few nuclear body-type structures (J.M. Bridger, H. Herrmann, C. Münkel, P. Lichter, J. Cell Sci., 111, 1241-1253), the distribution is entirely changed upon coupling with the evolutionarily conserved domain of the lamin B tail, the entire lamin B tail, the amino-terminal nucleoplasmic segment of the lamin B receptor (LBR), and the LEM domain of emerin, respectively. Remarkably, every individual chimeric protein exhibits a completely different distribution. Therefore, we assume that the chimeric parts are specifically recognized by factors engaged in nucleus-specific topogenesis. Thus, the conserved domain of the lamin B tail results in the formation of many small accumulations spread all over the nucleus. The chimera with the complete lamin B tail is deposited in short fibrillar aggregates within the nucleus. It does not mediate the integration of the chimeric protein into the nuclear membrane in cultured cells, indicating that the lamin tail alone is not sufficient to direct the integration of a protein into the lamina in vivo. In contrast, in the nuclear assembly system of Xenopus laevis the recombinant NLS-vimentin-lamin tail protein is concentrated at the nuclear membrane. The LBR chimera is arranged in a "beaded-chain"-type fashion, quite different from the more random deposition of NLS-vimentin alone. To our surprise, the LEM domain of emerin induces the retention of most of the chimeric proteins within the cytoplasm. Hence, it appears to be engaged in a strong cytoplasmic interaction that overrides the nuclear localization signal. Finally, the lamin chimera with the conserved part of the lamin B tail is shown to recruit LBR to the nuclear vimentin bodies and, vice versa, the LBR chimera attracts lamin B in transfected cells, thereby demonstrating their bona fide interaction in vivo.

Animals↗