QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online May 28, 2005
doi: 10.1242/10.1242/jcs.02376

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Brackmann, M. Articles by Braunewell, K.-H. Search for Related Content PubMed PubMed Citation Articles by Brackmann, M. Articles by Braunewell, K.-H. Social Bookmarking                         What's this?

Neuronal Ca²⁺ sensor protein VILIP-1 affects cGMP signalling of guanylyl cyclase B by regulating clathrin-dependent receptor recycling in hippocampal neurons

¹ Signal Transduction Research Group, Charité, University Medicine, 10117 Berlin, Germany
² Neuroscience Research Center, Charité, University Medicine, 10117 Berlin, Germany
³ Neuroscience Center of Excellence and Department of Neurology, Louisiana State University Health Sciences Center, New Orleans, LA, 70112, USA

View larger version (19K): [in a new window]   Fig. 1. (A) Enhancement of membrane cGMP levels following CNP-stimulation of natriuretic peptide receptor GC-B in untransfected and VILIP-1-transfected neural C6 cells. In VILIP-1-transfected glioma cells (C6-V1) and non-transfected C6 control cells (C6) cGMP accumulation was measured in unstimulated (Ctr) and CNP-stimulated (CNP, 0.2 µM) cells in the presence of the PDE inhibitor IBMX. (B) Time course of cGMP accumulation in non-transfected and VILIP-1-transfected C6 cells. Kinetic analysis of cGMP accumulation of non-transfected C6 cells (C6) and VILIP-1-transfected C6 cells (C6-V1) after CNP stimulation for various time points in the presence of IBMX. (C) Comparison of membrane cGMP levels following CNP-stimulation of natriuretic peptide receptor GC-B in untransfected, wild-type VILIP-1-, and myristoylation mutant-VILIP-1-transfected neural C6 cells. In wild-type VILIP-1-transfected glioma cells (C6-V1), myristoylation mutant VILIP-1-transfected (C6-V1M) and nontransfected C6 control cells (C6) cGMP accumulation was measured in unstimulated (Ctr) and CNP-stimulated (CNP, 0.2 µM) cells in the presence of the PDE inhibitor IBMX. (D) Expression of VILIP-1 and GC-B in non-transfected C6 and VILIP-1-transfected C6-V1 cells. Western blot analysis shows that no VILIP-1 expression can be detected in non-transfected C6 cells (lane 1), but can be detected as a 22 kDa protein in stably transfected C6-V1 cells (lane 2). The amount of GC-B protein with a Mr of 130 kDa (lanes 3, 4) does not differ between C6 cells (lane 3) and C6-V1 cells (lane 4) when compared with tubulin (50 kDa) as control. Mean values±s.d. are from two experiments for A and one representative experiment out of two for B and C carried out in triplicate.

View larger version (17K): [in a new window]   Fig. 2. (A) Surface expression of receptor GCs as shown by binding of biotinylated CNP in non-transfected and VILIP-1-transfected C6 cells. Non-transfected (C6) and VILIP-1-transfected (C6-V1) C6 cells were incubated with biotinylated CNP (0.2 µM for 20 seconds) and the relative surface expression of CNP-bound GC was measured using a streptavidin ELISA assay. (B) Time course of surface expression of GC receptors following ligand stimulation with biotinylated CNP in non-transfected and VILIP-1-transfected C6 cells. Non-transfected (C6) and VILIP-1-transfected (C6-V1) C6 cells were incubated with biotinylated CNP (0.2 µM) for a time period of 1, 5, 10, 15, 20 and 30 minutes and the surface expression of CNP-bound GC was measured in a streptavidin ELISA. Values were normalized to the 1 minutes value in C6 and C6-V1 cells, respectively. Mean values±s.e.m. are from five experiments in A and three in B. Asterisks (*,***) mark significant differences (P<0.05 with n=3, P<0.005 with n=5, Student's t-test).

View larger version (67K): [in a new window]   Fig. 3. Subcellular distribution of clathrin in non-transfected and VILIP-1-transfected C6 cells following CNP-stimulation as a measurement for membrane transport of GC receptor. (A) Western blot analysis using clathrin antibodies was performed with (+CNP) or without (–CNP) previous CNP-stimulation of non-transfected (C6) and VILIP-1-transfected (C6-V1) C6 cells and after separation of the P2 membrane fraction from the cytosolic fraction. (B) Quantification of the subcellular distribution of clathrin in non-transfected and VILIP-1-transfected C6 cells following CNP-stimulation. In non-transfected (C6) and VILIP-1-transfected (C6-V1) cells the association of clathrin with the P2 membrane fraction following CNP-stimulation was quantified using western blot analysis as shown in A. Mean values±s.e.m. are from seven experiments in B. Asterisks (*) mark a significant difference (P<0.05, n=7, Student's t-test). (C-F) Effect of GFP or VILIP-1-GFP expression on the subcellular localization of the transferrin receptor. C6 cells were transfected with GFP (E,F) or VILIP-1-GFP (C,D) and incubated with Rhodamine-transferrin for 13 minutes. After fixation the subcellular localization of Rhodamine-labelled transferrin (C-F, red channel) was observed by fluorescence microscopy and compared with VILIP-1-GFP (D, compare red and green channel) and GFP-expression (F, compare red and green channel). Bar in C=40 µm.

View larger version (94K): [in a new window]   Fig. 4. Co-localization of GC-B and endogenous VILIP-1 in hippocampal neurons. Hippocampal cell cultures (14 days in vitro) were labelled by indirect immunofluorescence using affinity-purified rat and rabbit polyclonal antibodies against VILIP-1 and GC-B, monoclonal MAP-2 and secondary Cy3 antibodies (red) or secondary Alexa FluorTM 488 antibodies (green). (A) Co-localization of VILIP-1 (red) and MAP-2 (green). (B) Co-localization of GC-B (red) and MAP-2 (green). Note, some MAP-2-positive neurons in A and B do not express VILIP-1 or GC-B (arrows). (C) Co-localization of VILIP-1 (green) and GC-B (red). (D) Magnification of hippocampal neurons in C expressing GC-B (red). (E) Magnification of hippocampal neurons in C expressing VILIP-1 (green). (F) Merged images of D and E show co-localization of GC-B and VILIP-1 (yellow) at higher magnification. (G) Localization of GC-B (red) in dendrites of a hippocampal neuron. (H) Localization of VILIP-1 (green) in dendrites of a hippocampal neuron. (I) Merged images of G and H show co-localization (yellow) of GC-B with VILIP-1 in hippocampal dendrites. Insets in D and E show background staining with secondary antibodies only. Bars in A=30 µm, in D=20 µm and in G=5 µm.

View larger version (17K): [in a new window]   Fig. 5. Protein expression of GC-B and of endogenous and heterologously expressed VILIP-1 in hippocampal neurons. (A) Homogenates of hippocampal cells were analyzed by western blot analysis using specific antibodies against VILIP-1 (lanes 1, 3 and 4) and GC-B (lanes 2, 5 and 6). VILIP-1-expression is shown in untransfected (lane 1), control-transfected (lane 3, pOPR) and VILIP-1-transfected (lane 4, pOPR-V1) hippocampal neurons. GC-B-expression is shown in untransfected (lane 2), control-transfected (lane 5, pOPR) and VILIP-1-transfected (lane 6, pOPR-V1) hippocampal neurons. Sizes of marker proteins are indicated at the left margin. 30 µg of protein was loaded in lanes 1 and 2, 10 µg in lanes 3-6. (B) Enhancement of membrane cGMP levels following CNP-stimulation of natriuretic receptor GC-B in untransfected and VILIP-1-transfected hippocampal neurons. CNP-stimulated cGMP accumulation in hippocampal neurons transiently transfected with control vector (pOPR) and vector containing VILIP-1-cDNA (pOPR-V1). Mean values±s.d. are from seven experiments carried out in triplicate. Asterisks (***) mark a significant difference between CNP-stimulated pOPR and pOPR-V1-transfected cells (P<0.005, n=7, Student's t-test). (C) Effect of different inhibitors of membrane transport on CNP-stimulated cGMP accumulation in control-transfected and VILIP-1-transfected hippocampal neurons. Control-transfected (pOPR) and VILIP-1-transfected (pOPR-V1) hippocampal neurons were stimulated with CNP for 20 minutes in the presence or absence of monensin (+Mon) or wortmannin (+WM), which inhibit receptor recycling at different stages; or in the presence of the endocytosis inhibitor PAO (+PAO). CNP-stimulated cGMP accumulation in control-transfected (pOPR) hippocampal neurons was set to 100% and all other values were expressed as % of control (CNP, pOPR). Mean values±s.d. are from at least three experiments, except PAO (two experiments), and were carried out in triplicate. Asterisks (*,***) mark significant differences (P<0.05, P<0.005, n=<3, Student's t-test).

View larger version (56K): [in a new window]   Fig. 6. Effect of heterologous VILIP-1 and VILIP-3 expression on the subcellular localization of GC-B protein in hippocampal neurons with and without CNP-stimulation. Hippocampal cell cultures (24 hours after transfection) were analyzed by confocal immunofluorescence microscopy using affinity-purified polyclonal antibodies against GC-B and secondary Cy3 anti-rabbit antibodies (red). GFP, VILIP-1-GFP and VILIP-3-GFP expression (green) was detected as green fluorescence of the GFP-fusion protein following transient transfection of the vector constructs. Subcellular localization of GC-B (red) was detected with (A,C,E) or without CNP-stimulation (B,D,F) in GFP-transfected (A,B), VILIP-1-GFP-transfected (C,D) and VILIP-3-GFP-transfected (E,F) hippocampal neurons. Upper insets show co-localization of GC-B with VILIP-1-GFP, VILIP-3-GFP or GFP expression (A-F, green). The lower insets in A-F show the quantification of the pixel values (relative GC-B fluorescence intensity) from a cross section of the shown neurons as indicated with the yellow bars. Black bars in the insets indicate the localization of the cell surface membrane. Bar in A=20 µm.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter