Mutations at Critical N-Glycosylation Sites Reduce Tyrosinase Activity by Altering Folding and Quality Control

A great majority of G protein-coupled receptors are modified by N-glycosylation, but the functional significance of this modification for receptor folding and intracellular transport has remained elusive. Here we studied these phenomena by mutating the two N-terminal N-glycosylation sites (Asn 18 and Asn 33 ) of the human ␦-opioid receptor, and expressing the mutants from the same chromosomal integration site in stably transfected inducible HEK293 cells. Both N-glycosylation sites were used, and their abolishment decreased the steady-state level of receptors at the cell surface. However, pulse-chase labeling, cell surface biotinylation, and immunofluorescence microscopy revealed that this was not because of intracellular accumulation. Instead, the non-N-glycosylated receptors were exported from the endoplasmic reticulum with enhanced kinetics. The results also revealed differences in the significance of the individual N-glycans, as the one attached to Asn 33 was found to be more important for endoplasmic reticulum retention of the receptor. The non-N-glycosylated receptors did not show gross functional impairment, but flow cytometry revealed that a fraction of them was incapable of ligand binding at the cell surface. In addition, the receptors that were devoid of N-glycans showed accelerated turnover and internalization and were targeted for lysosomal degradation. The results accentuate the importance of protein conformation-based screening before export from the endoplasmic reticulum, and demonstrate how the system is compromised when N-glycosylation is disrupted. We conclude that N-glycosylation of the ␦-opioid receptor is needed to maintain the expression of fully functional and stable receptor molecules at the cell surface.

Section: Discussionmentioning

confidence: 99%

N-Glycan-mediated Quality Control in the Endoplasmic Reticulum Is Required for the Expression of Correctly Folded δ-Opioid Receptors at the Cell Surface

Markkanen

Petäjä-Repo

2008

“…Only post-translationally the polypeptide chain reaches the native conformation, characterized by the presence of DTT-resistant disulfides. It has been documented that in the absence of CNX or specific N-glycans tyrosinase displays a nonproductive folding pathway (7,14). Similarly, the truncated chain is arrested in the co-translationally acquired non-native conformation.…”

Section: Discussionmentioning

confidence: 99%

Productive Folding of Tyrosinase Ectodomain Is Controlled by the Transmembrane Anchor

Popescu

Mares

Zdrentu

et al. 2006

Self Cite

Transmembrane domains (TMDs) are known as structural elements required for the insertion into the membrane of integral membrane proteins. We have provided here an example showing that the presence of the TMD is compulsory for the productive folding pathway of a membrane-anchored glycoprotein. Tyrosinase, a type I transmembrane protein whose insertion into the melanosomal membrane initiates melanin synthesis, is misfolded and degraded when expressed as a truncated polypeptide. We used constructs of tyrosinase ectodomain fused with chimeric TMDs or glycosylphosphatidylinositol anchor to gain insights into how the TMD enables the productive folding pathway of the ectodomain. We found that in contrast to the soluble constructs, the membrane-anchored chimeras fold into the native conformation, which allows their endoplasmic reticulum exit. They recruit calnexin to monitor their productive folding pathway characterized by the posttranslational formation of buried disulfides. Lacking calnexin assistance, the truncated mutant is arrested in an unstable conformation bearing exposed disulfides. We showed that the transmembrane anchor of a protein may crucially, albeit indirectly, control the folding pathway of the ectodomain.

“…The oligosaccharide moieties of a protein can affect its conformation, stability, and/or transport in cells (36,37). For all membrane-bound ACs, potential N-linked glycosylation sites are located on the predicted extracellular loop 5 and/or loop 6 ( Table I).…”

Section: Discussionmentioning

confidence: 99%

N-Glycosylation and Residues Asn805 and Asn890 Are Involved in the Functional Properties of Type VI Adenylyl Cyclase

Wu¹,

Lai²,

Lin³

et al. 2001

In this study, we demonstrate that type VI adenylyl cyclase (ACVI) is glycosylated in vivo. Treating HEK293 cells expressing ACVI with tunicamycin to block the addition of N-linked oligosaccharide or removing the N-linked oligosaccharide by in vitro peptidyl-N-glycosidase F digestion reduced the molecular mass of ACVI. Furthermore, tunicamycin treatment suppressed the forskolin-stimulated activity of ACVI. Mutation of either one or both potential N-glycosylation sites (Asn 805 and Asn 890 , located on extracellular loops 5 and 6, respectively) also reduced the molecular mass of ACVI. Therefore, ACVI was glycosylated at both Asn 805 and Asn 890 . Confocal analysis indicated that glycosylation was not required for the delivery of ACVI to the cell surface. Although no significant alterations in K m values for ATP or sensitivity to divalent cations were detected, the glycosylation-deficient ACVI mutant N805Q/N890Q-ACVI exhibited much lower forskolin-, Mn 2؉ -, and Mg 2؉ -stimulated cyclase activities than did wild-type ACVI. By contrast, the G␣ s -stimulated cyclase activities of wildtype ACVI and N805Q/N890Q-ACVI were indistinguishable. Furthermore, compared with wild-type ACVI, N805Q/N890Q-ACVI was less sensitive to inhibition mediated by dopamine D2 receptors or by protein kinase C. Collectively, glycosylation of ACVI not only affected its catalytic activity in an activator-dependent manner, but also altered its ability to be regulated by a G␣ i proteincoupled receptor or by protein kinase C. Adenylyl cyclases (ACs)1 produce cAMP in response to extracellular stimulation. To date, at least nine mammalian membrane-bound ACs have been isolated and characterized (1). All membrane-bound ACs contain two hydrophobic regions that comprise six transmembrane helices and three large cytoplasmic domains (N, C1a/b, and C2) (see Fig. 1). The basic catalytic unit is very conserved among ACs and is composed of the C1a and C2 domains (2-4). The ␣-subunit of the G s protein (G␣ s ), which activates all membrane-bound ACs, at least in vitro, was shown to interact with the catalytic complex of ACs at the C2 domain (5, 6). On the contrary, the ␣-subunit of the G i protein (G␣ i ) only effectively suppresses some of these membranebound AC isozymes (i.e. type I, V, and VI ACs) (7). The G␣ i protein was reported to interact with the C1 domain of type V AC (3). The N-terminal domains of ACs are highly variable and were shown to play modulating roles (9, 10). A recent study by Chen et al. (11) suggests that the two tandem six-transmembrane segments are critical for the targeting and functional assembly of ACs. The regulatory roles of the transmembrane domains and of the six extracellular loops of ACs remain largely uncharacterized.Members of the AC superfamily are tightly controlled by various signals and therefore contribute significantly to the complexity and fine-tuning of cellular signaling mechanisms (12). We are particularly interested in type VI AC (ACVI) because we (9, 13) and others (14, 15) have shown that ACVI can be regulated...