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Retina specific GCAPs in zebrafish acquire functional selectivity in Ca2+-sensing by myristoylation and Mg2+-binding.

Sulmann S, Vocke F, Scholten A, Koch KW - Sci Rep (2015)

Bottom Line: Myristoylation does not facilitate membrane binding of zGCAPs, but it significantly modified the regulatory properties of zGCAP2 and zGCAP5.Hydrodynamic properties of zGCAPs are more influenced by Ca(2+) than by Mg(2+), although to a different extent for each zGCAP.Posttranslational modification and competing ion-binding can tailor the properties of similar Ca(2+)-sensors.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosciences, Biochemistry Group, University of Oldenburg, D-26111-Oldenburg, Germany.

ABSTRACT
Zebrafish photoreceptor cells express six guanylate cyclase-activating proteins (zGCAPs) that share a high degree of amino acid sequence homology, but differ in Ca(2+)-binding properties, Ca(2+)-sensitive target regulation and spatial-temporal expression profiles. We here study a general problem in cellular Ca(2+)-sensing, namely how similar Ca(2+)-binding proteins achieve functional selectivity to control finely adjusted cellular responses. We investigated two parameters of critical importance for the trigger and switch function of guanylate cyclase-activating proteins: the myristoylation status and the occupation of Ca(2+)-binding sites with Mg(2+). All zGCAPs can be myristoylated in living cells using click chemistry. Myristoylation does not facilitate membrane binding of zGCAPs, but it significantly modified the regulatory properties of zGCAP2 and zGCAP5. We further determined for all zGCAPs at least two binding sites exhibiting high affinities for Ca(2+) with KD values in the submicromolar range, whereas for other zGCAPs (except zGCAP3) the affinity of the third binding site was in the micromolar range. Mg(2+) either occupied the low affinity Ca(2+)-binding site or it shifted the affinities for Ca(2+)-binding. Hydrodynamic properties of zGCAPs are more influenced by Ca(2+) than by Mg(2+), although to a different extent for each zGCAP. Posttranslational modification and competing ion-binding can tailor the properties of similar Ca(2+)-sensors.

No MeSH data available.


Related in: MedlinePlus

Acylation of zGCAPs in living cells by double fluorescence detection.HEK 293 cells were transfected with GFP constructs of zGCAPs and recoverin, which were acylated in vivo and labeled with the DIBO-TAMRA dye. Nuclear DAPI staining is displayed in left most column (a,e,i,m,q,u), GFP fluorescence in second left column (b,f,j,n,r,v), DIBO-TAMRA fluorescence in the third column (c,g,k,o) and an overlay of all signals in fourth column (d,h,l,p,t,x). GFP and DIBO-TAMRA fluorescence mainly overlaps for cytoplasmic regions. Pictures in panels s and w (column three) are bright field images of HEK cells that were not treated with the azido myristic acid substitute. Mock transfected cells are shown as controls. Figures were taken by using the LUCP PlanFi 40 x/0.60 olympus objective and the DAPI/Fitc/TexasRed Filter Set (Olympus). Scale bars: 20 μm.
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f2: Acylation of zGCAPs in living cells by double fluorescence detection.HEK 293 cells were transfected with GFP constructs of zGCAPs and recoverin, which were acylated in vivo and labeled with the DIBO-TAMRA dye. Nuclear DAPI staining is displayed in left most column (a,e,i,m,q,u), GFP fluorescence in second left column (b,f,j,n,r,v), DIBO-TAMRA fluorescence in the third column (c,g,k,o) and an overlay of all signals in fourth column (d,h,l,p,t,x). GFP and DIBO-TAMRA fluorescence mainly overlaps for cytoplasmic regions. Pictures in panels s and w (column three) are bright field images of HEK cells that were not treated with the azido myristic acid substitute. Mock transfected cells are shown as controls. Figures were taken by using the LUCP PlanFi 40 x/0.60 olympus objective and the DAPI/Fitc/TexasRed Filter Set (Olympus). Scale bars: 20 μm.

Mentions: In a second alternative approach we employed a copperless cycloaddition suitable for introducing a fluorescent dye (DIBO-TAMRA-dye) to the fatty acyl group of the NCS proteins in living cells. Thus we were able to colocalize the putative N-terminal attached fatty acyl chain and the C-terminal attached GFP to the protein in transfected cells. Figure 2 gives an overview of the results obtained with fluorescence microscopy for recoverin, zGCAP3 and 5. GFP and TAMRA fluorescence mainly overlapped for cytoplasmic regions indicating the presence of a covalently attached acylgroup on fluorescently labeled zGCAP3 and 5 (Fig. 2a–h). A similar localization pattern was observed with the other zGCAPs (data not shown). Cells expressing GFP-labelled zGCAP3 and recoverin (Fig. 2q–x) that were not modified by the azido-modified myristic acid substitute appear normal in shape comparable to previous results obtained with mammalian GFP-labelled GCAPs1920. However, we observed no uniform spreading to the nucleus. Further, when we added the myristic acid substitute allowing the subsequent cycloaddition with the DIBO-TAMRA dye cell shape was affected leading to the round form visible in Fig. 2a–l (see also Figure S1 in supplement). Myristoylated recoverin was found in the vicinity to membranes, but it was also detected in restricted cytosolic regions (Fig. 2j–l and r–t). This might indicate partial association of recoverin with membrane structures at low Ca2+-concentration, which had been observed in previous studies and is mainly due to hydrophobic/ electrostatic interactions212223.


Retina specific GCAPs in zebrafish acquire functional selectivity in Ca2+-sensing by myristoylation and Mg2+-binding.

Sulmann S, Vocke F, Scholten A, Koch KW - Sci Rep (2015)

Acylation of zGCAPs in living cells by double fluorescence detection.HEK 293 cells were transfected with GFP constructs of zGCAPs and recoverin, which were acylated in vivo and labeled with the DIBO-TAMRA dye. Nuclear DAPI staining is displayed in left most column (a,e,i,m,q,u), GFP fluorescence in second left column (b,f,j,n,r,v), DIBO-TAMRA fluorescence in the third column (c,g,k,o) and an overlay of all signals in fourth column (d,h,l,p,t,x). GFP and DIBO-TAMRA fluorescence mainly overlaps for cytoplasmic regions. Pictures in panels s and w (column three) are bright field images of HEK cells that were not treated with the azido myristic acid substitute. Mock transfected cells are shown as controls. Figures were taken by using the LUCP PlanFi 40 x/0.60 olympus objective and the DAPI/Fitc/TexasRed Filter Set (Olympus). Scale bars: 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4462140&req=5

f2: Acylation of zGCAPs in living cells by double fluorescence detection.HEK 293 cells were transfected with GFP constructs of zGCAPs and recoverin, which were acylated in vivo and labeled with the DIBO-TAMRA dye. Nuclear DAPI staining is displayed in left most column (a,e,i,m,q,u), GFP fluorescence in second left column (b,f,j,n,r,v), DIBO-TAMRA fluorescence in the third column (c,g,k,o) and an overlay of all signals in fourth column (d,h,l,p,t,x). GFP and DIBO-TAMRA fluorescence mainly overlaps for cytoplasmic regions. Pictures in panels s and w (column three) are bright field images of HEK cells that were not treated with the azido myristic acid substitute. Mock transfected cells are shown as controls. Figures were taken by using the LUCP PlanFi 40 x/0.60 olympus objective and the DAPI/Fitc/TexasRed Filter Set (Olympus). Scale bars: 20 μm.
Mentions: In a second alternative approach we employed a copperless cycloaddition suitable for introducing a fluorescent dye (DIBO-TAMRA-dye) to the fatty acyl group of the NCS proteins in living cells. Thus we were able to colocalize the putative N-terminal attached fatty acyl chain and the C-terminal attached GFP to the protein in transfected cells. Figure 2 gives an overview of the results obtained with fluorescence microscopy for recoverin, zGCAP3 and 5. GFP and TAMRA fluorescence mainly overlapped for cytoplasmic regions indicating the presence of a covalently attached acylgroup on fluorescently labeled zGCAP3 and 5 (Fig. 2a–h). A similar localization pattern was observed with the other zGCAPs (data not shown). Cells expressing GFP-labelled zGCAP3 and recoverin (Fig. 2q–x) that were not modified by the azido-modified myristic acid substitute appear normal in shape comparable to previous results obtained with mammalian GFP-labelled GCAPs1920. However, we observed no uniform spreading to the nucleus. Further, when we added the myristic acid substitute allowing the subsequent cycloaddition with the DIBO-TAMRA dye cell shape was affected leading to the round form visible in Fig. 2a–l (see also Figure S1 in supplement). Myristoylated recoverin was found in the vicinity to membranes, but it was also detected in restricted cytosolic regions (Fig. 2j–l and r–t). This might indicate partial association of recoverin with membrane structures at low Ca2+-concentration, which had been observed in previous studies and is mainly due to hydrophobic/ electrostatic interactions212223.

Bottom Line: Myristoylation does not facilitate membrane binding of zGCAPs, but it significantly modified the regulatory properties of zGCAP2 and zGCAP5.Hydrodynamic properties of zGCAPs are more influenced by Ca(2+) than by Mg(2+), although to a different extent for each zGCAP.Posttranslational modification and competing ion-binding can tailor the properties of similar Ca(2+)-sensors.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosciences, Biochemistry Group, University of Oldenburg, D-26111-Oldenburg, Germany.

ABSTRACT
Zebrafish photoreceptor cells express six guanylate cyclase-activating proteins (zGCAPs) that share a high degree of amino acid sequence homology, but differ in Ca(2+)-binding properties, Ca(2+)-sensitive target regulation and spatial-temporal expression profiles. We here study a general problem in cellular Ca(2+)-sensing, namely how similar Ca(2+)-binding proteins achieve functional selectivity to control finely adjusted cellular responses. We investigated two parameters of critical importance for the trigger and switch function of guanylate cyclase-activating proteins: the myristoylation status and the occupation of Ca(2+)-binding sites with Mg(2+). All zGCAPs can be myristoylated in living cells using click chemistry. Myristoylation does not facilitate membrane binding of zGCAPs, but it significantly modified the regulatory properties of zGCAP2 and zGCAP5. We further determined for all zGCAPs at least two binding sites exhibiting high affinities for Ca(2+) with KD values in the submicromolar range, whereas for other zGCAPs (except zGCAP3) the affinity of the third binding site was in the micromolar range. Mg(2+) either occupied the low affinity Ca(2+)-binding site or it shifted the affinities for Ca(2+)-binding. Hydrodynamic properties of zGCAPs are more influenced by Ca(2+) than by Mg(2+), although to a different extent for each zGCAP. Posttranslational modification and competing ion-binding can tailor the properties of similar Ca(2+)-sensors.

No MeSH data available.


Related in: MedlinePlus