PMID- 10497248
OWN - NLM
STAT- MEDLINE
DCOM- 19991102
LR  - 20210209
IS  - 0021-9258 (Print)
IS  - 0021-9258 (Linking)
VI  - 274
IP  - 40
DP  - 1999 Oct 1
TI  - The N-terminal anchor sequences of 11beta-hydroxysteroid dehydrogenases determine
      their orientation in the endoplasmic reticulum membrane.
PG  - 28762-70
AB  - 11beta-Hydroxysteroid dehydrogenase enzymes (11beta- HSD) regulate the ratio of
      active endogenous glucocorticoids to their inactive keto-metabolites, thereby
      controlling the access of glucocorticoids to their cognate receptors. In this
      study, the topology and intracellular localization of 11beta-HSD1 and 11beta-HSD2
      have been analyzed by immunohistochemistry and protease protection assays of in
      vitro transcription/translation products. 11beta-HSD constructs, tagged with the 
      FLAG epitope, were transiently expressed in HEK-293 cells. The enzymatic
      characteristics of tagged and native enzymes were indistinguishable. Fluorescence
      microscopy demonstrated the localization of both 11beta-HSD1 and 11beta-HSD2
      exclusively to the endoplasmic reticulum (ER) membrane. To examine the
      orientation of tagged 11beta-HSD enzymes within the ER membrane, we stained
      selectively permeabilized HEK-293 cells with anti-FLAG antibody.
      Immunohistochemistry revealed that the N terminus of 11beta-HSD1 is cytoplasmic, 
      and the catalytic domain containing the C terminus is protruding into the ER
      lumen. In contrast, the N terminus of 11beta-HSD2 is lumenal, and the catalytic
      domain is facing the cytoplasm. Chimeric proteins where the N-terminal anchor
      sequences of 11beta-HSD1 and 11beta-HSD2 were exchanged adopted inverted
      orientation in the ER membrane. However, both chimeric proteins were not
      catalytically active. Furthermore, mutation of a tyrosine motif to alanine in the
      transmembrane segment of 11beta-HSD1 significantly reduced V(max). The
      subcellular localization of 11beta-HSD1 was not affected by mutations of the
      tyrosine motif or of a di-lysine motif in the N terminus. However, residue
      Lys(5), but not Lys(6), turned out to be critical for the topology of
      11beta-HSD1. Mutation of Lys(5) to Ser inverted the orientation of 11beta-HSD1 in
      the ER membrane without loss of catalytic activity. Our results emphasize the
      importance of the N-terminal transmembrane segments of 11beta-HSD enzymes for
      their proper function and demonstrate that they are sufficient to determine their
      orientation in the ER membrane.
FAU - Odermatt, A
AU  - Odermatt A
AD  - Division of Nephrology, Department of Medicine, University of Berne, 3010 Berne, 
      Switzerland. alex.odermatt@dkf2.unibe.ch
FAU - Arnold, P
AU  - Arnold P
FAU - Stauffer, A
AU  - Stauffer A
FAU - Frey, B M
AU  - Frey BM
FAU - Frey, F J
AU  - Frey FJ
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - J Biol Chem
JT  - The Journal of biological chemistry
JID - 2985121R
RN  - 0 (DNA Primers)
RN  - 0 (Isoenzymes)
RN  - EC 1.1.- (Hydroxysteroid Dehydrogenases)
RN  - EC 1.1.1.146 (11-beta-Hydroxysteroid Dehydrogenases)
SB  - IM
MH  - 11-beta-Hydroxysteroid Dehydrogenases
MH  - Amino Acid Sequence
MH  - Base Sequence
MH  - Catalysis
MH  - DNA Primers
MH  - Endoplasmic Reticulum/*enzymology
MH  - Humans
MH  - Hydroxysteroid Dehydrogenases/chemistry/genetics/*metabolism
MH  - Intracellular Membranes/enzymology
MH  - Isoenzymes/chemistry/genetics/*metabolism
MH  - Kinetics
MH  - Mutagenesis
EDAT- 1999/09/25 00:00
MHDA- 1999/09/25 00:01
CRDT- 1999/09/25 00:00
PHST- 1999/09/25 00:00 [pubmed]
PHST- 1999/09/25 00:01 [medline]
PHST- 1999/09/25 00:00 [entrez]
AID - 10.1074/jbc.274.40.28762 [doi]
AID - S0021-9258(19)52111-1 [pii]
PST - ppublish
SO  - J Biol Chem. 1999 Oct 1;274(40):28762-70. doi: 10.1074/jbc.274.40.28762.