A primate-dominant third glycosylation site of the β2- adrenergic receptor routes receptors to degradation during agonist regulation

Abstract

β2-adrenergic receptors (β2AR) of all species are N-linked glycosylated at amino terminus residues ∼6 and ∼15. However, the human β2AR has a potential third N-glycosylation site at ECL2 residue 187. To determine whether this residue is glycosylated and to ascertain function, all possible single/multiple Asn → Gln mutations were made in the human β2AR at positions 6, 15, and 187 and were expressed in Chinese hamster fibroblast cells. Substitution of Asn-187 alone or with Asn-6 or Asn-15 decreased the apparent molecular mass of the receptor on SDS-PAGE in a manner consistent with Asn-187 glycosylation. All receptors bound the agonist isoproterenol and functionally coupled to adenylyl cyclase. However, receptors without 187 glycosylation failed to display long term agonist-promoted down-regulation. In contrast, loss of Asn-6/Asn-15 glycosylation did not alter down-regulation. Cell surface distribution and agonist-promoted internalization of receptors and recruitment of β-arrestin 2 were unaffected by the loss of 187 glycosylation. Furthermore, acutely internalized wild-type and Gln-187 receptors were both localized by confocal microscopy to early endosomes. During prolonged agonist exposure, wild-type β2AR co-localized with lysosomes, consistent with trafficking to a degradation compartment. However, Gln-187 β2AR failed to co-localize with lysosomes despite agonist treatments up to 18 h. Phylogenetic analysis revealed that this third glycosylation site is found in humans and other higher order primates but not in lower order primates such as the monkey. Nor is this third site found in rodents, which are frequently utilized as animal models. These data thus reveal a previously unrecognized β2AR regulatory motif that appeared late in primate evolution and serves to direct internalized receptors to lysosomal degradation during long term agonist exposure.