Human G protein-coupled receptor 30 is N-glycosylated and N-terminal domain asparagine 44 is required for receptor structure and activity.

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 N-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, 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 approximately 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 role(s).