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Human G protein-coupled Receptor 30 (GPR30) is N -glycosylated and N-terminal Domain Asparagine 44 is Required for Receptor Structure and Activity

Gonzalez de Valdivia, Ernesto LU ; Sandén, Caroline LU ; Kahn, Robin LU ; Olde, Björn LU and Leeb-Lundberg, Fredrik L M LU (2019) In Bioscience Reports 39(2).
Abstract

GPR30, or G protein-coupled estrogen receptor (GPER), is a G protein-coupled receptor (GPCR) that is currently attracting considerable attention in breast cancer and cardiometabolic regulation. The receptor was reported to be a novel membrane estrogen receptor mediating rapid non-genomic responses. However, questions remain about both the cognate ligand and the subcellular localization of receptor activity. Here, we used HEK293 cells ectopically expressing N-terminally FLAG-tagged human GPR30 and three unique antibodies (Ab) specifically targeting the receptor N-terminal domain (N-domain) to investigate the role of N -glycosylation in receptor maturation and activity, the latter assayed by constitutive receptor-stimulated ERK1/2... (More)

GPR30, or G protein-coupled estrogen receptor (GPER), is a G protein-coupled receptor (GPCR) that is currently attracting considerable attention in breast cancer and cardiometabolic regulation. The receptor was reported to be a novel membrane estrogen receptor mediating rapid non-genomic responses. However, questions remain about both the cognate ligand and the subcellular localization of receptor activity. Here, we used HEK293 cells ectopically expressing N-terminally FLAG-tagged human GPR30 and three unique antibodies (Ab) specifically targeting the receptor N-terminal domain (N-domain) to investigate the role of N -glycosylation in receptor maturation and activity, the latter assayed by constitutive receptor-stimulated ERK1/2 activity. GPR30 expression was complex with receptor species spanning from about 40 kDa to higher molecular masses and localized in the endoplasmatic reticulum (ER), the plasma membrane (PM), and endocytic vesicles. The receptor contains three conserved asparagines, Asn25, Asn32, and Asn44, in consensus N -glycosylation motifs, all in the N-domain, and PNGase F treatment showed that at least one of them is N -glycosylated. Mutating Asn44 to isoleucine inactivated the receptor, yielding a unique receptor species at about 20 kDa that was recognized by Ab only in a denatured state. On the other hand, mutating Asn25 or Asn32 either individually or in combination, or truncating successively N-domain residues 1-42, had no significant effect either on receptor structure, maturation, or activity. Thus, Asn44 in the GPR30 N-domain is required for receptor structure and activity, whereas N-domain residues 1-42, including specifically Asn25 and Asn32, do not play any major structural or functional roles.

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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Bioscience Reports
volume
39
issue
2
publisher
Kluwer
external identifiers
  • scopus:85062708701
ISSN
0144-8463
DOI
10.1042/BSR20182436
language
English
LU publication?
yes
id
2c3635e0-55d7-4afa-a046-1e9a0da9fe57
date added to LUP
2019-02-18 14:30:50
date last changed
2019-04-23 04:46:03
@article{2c3635e0-55d7-4afa-a046-1e9a0da9fe57,
  abstract     = {<p>GPR30, or G protein-coupled estrogen receptor (GPER), is a G protein-coupled receptor (GPCR) that is currently attracting considerable attention in breast cancer and cardiometabolic regulation. The receptor was reported to be a novel membrane estrogen receptor mediating rapid non-genomic responses. However, questions remain about both the cognate ligand and the subcellular localization of receptor activity. Here, we used HEK293 cells ectopically expressing N-terminally FLAG-tagged human GPR30 and three unique antibodies (Ab) specifically targeting the receptor N-terminal domain (N-domain) to investigate the role of N -glycosylation in receptor maturation and activity, the latter assayed by constitutive receptor-stimulated ERK1/2 activity. GPR30 expression was complex with receptor species spanning from about 40 kDa to higher molecular masses and localized in the endoplasmatic reticulum (ER), the plasma membrane (PM), and endocytic vesicles. The receptor contains three conserved asparagines, Asn25, Asn32, and Asn44, in consensus N -glycosylation motifs, all in the N-domain, and PNGase F treatment showed that at least one of them is N -glycosylated. Mutating Asn44 to isoleucine inactivated the receptor, yielding a unique receptor species at about 20 kDa that was recognized by Ab only in a denatured state. On the other hand, mutating Asn25 or Asn32 either individually or in combination, or truncating successively N-domain residues 1-42, had no significant effect either on receptor structure, maturation, or activity. Thus, Asn44 in the GPR30 N-domain is required for receptor structure and activity, whereas N-domain residues 1-42, including specifically Asn25 and Asn32, do not play any major structural or functional roles.</p>},
  articleno    = {BSR20182436},
  author       = {Gonzalez de Valdivia, Ernesto and Sandén, Caroline and Kahn, Robin and Olde, Björn and Leeb-Lundberg, Fredrik L M},
  issn         = {0144-8463},
  language     = {eng},
  month        = {02},
  number       = {2},
  publisher    = {Kluwer},
  series       = {Bioscience Reports},
  title        = {Human G protein-coupled Receptor 30 (GPR30) is N -glycosylated and N-terminal Domain Asparagine 44 is Required for Receptor Structure and Activity},
  url          = {http://dx.doi.org/10.1042/BSR20182436},
  volume       = {39},
  year         = {2019},
}