Membrane localization of cyclic nucleotide phosphodiesterase 3 (PDE3). Two N-terminal domains are required for the efficient targeting to, and association of, PDE3 with endoplasmic reticulum
Shakur, Y ; Takeda, K ; Kenan, Y ; Yu, Z X ; Rena, G ; Brandt, D ; Houslay, M D ; Degerman, Eva LU
; Ferrans, V J
and Manganiello, V C
(2000)
In Journal of Biological Chemistry
275(49).
p.38749-38761
- Abstract
- Subcellular localization of cyclic nucleotide phosphodiesterases (PDEs) may be important in compartmentalization of cAMP/cGMP signaling responses. In 3T3-L1 adipocytes, mouse (M) PDE3B was associated with the endoplasmic reticulum (ER) as indicated by its immunofluorescent colocalization with the ER protein BiP and subcellular fractionation studies. In transfected NIH 3006 or COS-7 cells, recombinant wild-type PDE3A and PDE3B isoforms were both found almost exclusively in the ER. The N-terminal portion of PDE3 can be arbitrarily divided into region 1 (aa 1-300), which contains a large hydrophobic domain with six predicted transmembrane helices, followed by region 2 (aa 301-500) containing a smaller hydrophobic domain (of approximately 50... (More)
- Subcellular localization of cyclic nucleotide phosphodiesterases (PDEs) may be important in compartmentalization of cAMP/cGMP signaling responses. In 3T3-L1 adipocytes, mouse (M) PDE3B was associated with the endoplasmic reticulum (ER) as indicated by its immunofluorescent colocalization with the ER protein BiP and subcellular fractionation studies. In transfected NIH 3006 or COS-7 cells, recombinant wild-type PDE3A and PDE3B isoforms were both found almost exclusively in the ER. The N-terminal portion of PDE3 can be arbitrarily divided into region 1 (aa 1-300), which contains a large hydrophobic domain with six predicted transmembrane helices, followed by region 2 (aa 301-500) containing a smaller hydrophobic domain (of approximately 50 aa). To investigate the role of regions 1 and 2 in membrane association, we examined the subcellular localization of a series of catalytically active, Flag-tagged N-terminal-truncated human (H) PDE3A and MPDE3B recombinants, as well as a series of fragments from regions 1 and 2 of MPDE3B synthesized as enhanced green fluorescent (EGFP) fusion proteins in COS-7 cells. In COS-7 cells, the localization of a mutant HPDE3A, lacking the first 189 amino acids (aa) and therefore four of the six predicted transmembrane helices (H3A-Delta189), was virtually identical to that of the wild type. M3B-Delta302 (lacking region 1) and H3A-Delta397 (lacking region 1 as well as part of region 2) retained, to different degrees, the ability to associate with membranes, albeit less efficiently than H3A-Delta189. Proteins that lacked both regions 1 and 2, H3A-Delta510 and M3B-Delta604, did not associate with membranes. Consistent with these findings, region 1 EGFP-MPDE3B fusion proteins colocalized with the ER, whereas region 2 EGFP fusion proteins were diffusely distributed. Thus, some portion of the N-terminal hydrophobic domain in region 1 plus a second domain in region 2 are important for efficient membrane association/targeting of PDE3. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/1116404
- author
- Shakur, Y
; Takeda, K
; Kenan, Y
; Yu, Z X
; Rena, G
; Brandt, D
; Houslay, M D
; Degerman, Eva
LU
; Ferrans, V J
and Manganiello, V C
- organization
- publishing date
- 2000
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- Endoplasmic Reticulum, Localization, Cyclic Nucleotide Phosphodiesterase, Membrane-Association, Immunofluorescence
- in
- Journal of Biological Chemistry
- volume
- 275
- issue
- 49
- pages
- 38749 - 38761
- publisher
- American Society for Biochemistry and Molecular Biology
- external identifiers
-
- pmid:10952971
- scopus:17744398004
- pmid:10952971
- ISSN
- 1083-351X
- DOI
- 10.1074/jbc.M001734200
- language
- English
- LU publication?
- yes
- id
- 1101d389-78ba-4fc5-a977-dbfec0c96d91 (old id 1116404)
- date added to LUP
- 2016-04-01 12:34:47
- date last changed
- 2022-01-27 07:00:46
@article{1101d389-78ba-4fc5-a977-dbfec0c96d91,
abstract = {{Subcellular localization of cyclic nucleotide phosphodiesterases (PDEs) may be important in compartmentalization of cAMP/cGMP signaling responses. In 3T3-L1 adipocytes, mouse (M) PDE3B was associated with the endoplasmic reticulum (ER) as indicated by its immunofluorescent colocalization with the ER protein BiP and subcellular fractionation studies. In transfected NIH 3006 or COS-7 cells, recombinant wild-type PDE3A and PDE3B isoforms were both found almost exclusively in the ER. The N-terminal portion of PDE3 can be arbitrarily divided into region 1 (aa 1-300), which contains a large hydrophobic domain with six predicted transmembrane helices, followed by region 2 (aa 301-500) containing a smaller hydrophobic domain (of approximately 50 aa). To investigate the role of regions 1 and 2 in membrane association, we examined the subcellular localization of a series of catalytically active, Flag-tagged N-terminal-truncated human (H) PDE3A and MPDE3B recombinants, as well as a series of fragments from regions 1 and 2 of MPDE3B synthesized as enhanced green fluorescent (EGFP) fusion proteins in COS-7 cells. In COS-7 cells, the localization of a mutant HPDE3A, lacking the first 189 amino acids (aa) and therefore four of the six predicted transmembrane helices (H3A-Delta189), was virtually identical to that of the wild type. M3B-Delta302 (lacking region 1) and H3A-Delta397 (lacking region 1 as well as part of region 2) retained, to different degrees, the ability to associate with membranes, albeit less efficiently than H3A-Delta189. Proteins that lacked both regions 1 and 2, H3A-Delta510 and M3B-Delta604, did not associate with membranes. Consistent with these findings, region 1 EGFP-MPDE3B fusion proteins colocalized with the ER, whereas region 2 EGFP fusion proteins were diffusely distributed. Thus, some portion of the N-terminal hydrophobic domain in region 1 plus a second domain in region 2 are important for efficient membrane association/targeting of PDE3.}},
author = {{Shakur, Y and Takeda, K and Kenan, Y and Yu, Z X and Rena, G and Brandt, D and Houslay, M D and Degerman, Eva and Ferrans, V J and Manganiello, V C}},
issn = {{1083-351X}},
keywords = {{Endoplasmic Reticulum; Localization; Cyclic Nucleotide Phosphodiesterase; Membrane-Association; Immunofluorescence}},
language = {{eng}},
number = {{49}},
pages = {{38749--38761}},
publisher = {{American Society for Biochemistry and Molecular Biology}},
series = {{Journal of Biological Chemistry}},
title = {{Membrane localization of cyclic nucleotide phosphodiesterase 3 (PDE3). Two N-terminal domains are required for the efficient targeting to, and association of, PDE3 with endoplasmic reticulum}},
url = {{http://dx.doi.org/10.1074/jbc.M001734200}},
doi = {{10.1074/jbc.M001734200}},
volume = {{275}},
year = {{2000}},
}