Department of Experimental Medical Science, Lund University, Lund 22184, Sweden.
Department of Pediatrics, Lund University, Lund 22184, Sweden.
Biosci Rep. 2019 Feb 26;39(2). doi: 10.1042/BSR20182436. Print 2019 Feb 28.
G protein-coupled receptor 30 (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 human embryonic kidney (HEK) 293 (HEK293) cells ectopically expressing N-terminally FLAG-tagged human GPR30 and three unique antibodies (Ab) specifically targetting the receptor N-terminal domain (N-domain) to investigate the role of -glycosylation in receptor maturation and activity, the latter assayed by constitutive receptor-stimulated extracellular-regulated protein kinase (ERK) 1/2 (ERK1/2) activity. GPR30 expression was complex with receptor species spanning from approximately 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, Asn, Asn, and Asn, in consensus -glycosylation motifs, all in the N-domain, and PNGase F treatment showed that at least one of them is -glycosylated. Mutating Asn to isoleucine inactivated the receptor, yielding a unique receptor species at approximately 20 kDa that was recognized by Ab only in a denatured state. On the other hand, mutating Asn or Asn 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, Asn in the GPR30 N-domain is required for receptor structure and activity, whereas N-domain residues 1-42, including specifically Asn and Asn, do not play any major structural or functional role(s).
G 蛋白偶联受体 30（GPR30），又称 G 蛋白偶联雌激素受体（GPER），是一种 G 蛋白偶联受体（GPCR），目前在乳腺癌和心脏代谢调节方面引起了广泛关注。该受体被报道为一种新型的膜雌激素受体，介导快速的非基因组反应。然而，关于配体和受体活性的亚细胞定位问题仍然存在。在这里，我们使用人胚肾（HEK）293（HEK293）细胞中过表达的 N 端 FLAG 标记的人 GPR30 和三种针对受体 N 端结构域（N 结构域）的独特抗体（Ab），研究 - 糖基化在受体成熟和活性中的作用，后者通过组成型受体刺激细胞外调节蛋白激酶（ERK）1/2（ERK1/2）活性来测定。GPR30 的表达与受体物种复杂，跨度约为 40 kDa 至更高的分子量，并定位于内质网（ER）、质膜（PM）和内吞小泡中。该受体包含三个保守的天冬酰胺，Asn、Asn 和 Asn，在共识 - 糖基化基序中，都在 N 结构域中，并且 PNGase F 处理表明至少有一个是 - 糖基化的。将 Asn 突变为异亮氨酸使受体失活，产生约 20 kDa 的独特受体物种，仅在变性状态下被 Ab 识别。另一方面，将 Asn 或 Asn 单独或组合突变，或依次截短 N 结构域残基 1-42，对受体结构、成熟或活性均无显著影响。因此，GPR30 N 结构域中的 Asn 是受体结构和活性所必需的，而 N 结构域残基 1-42，包括特定的 Asn 和 Asn，不发挥任何主要的结构或功能作用。
