Palmitoylation and ubiquitination regulate exit of the Wnt signaling protein LRP6 from the endoplasmic reticulum

Abstract

Canonical Wnt signaling is initiated by binding of Wnt proteins to members of the Frizzled family and subsequent complex formation with lipoprotein receptor-related proteins 5/6 (LRP5/6). Here, we show that LRP6 is palmitoylated on a juxtamembranous cysteine and that palmitoylation is required for exit from the endoplasmic reticulum (ER). We propose that palmitoylation serves to tilt the long, 23-residue transmembrane domain of LRP6 with respect to the plane of membrane to prevent a hydrophobic mismatch and subsequent recognition by the ER quality control. In support of this model, a palmitoylation-deficient LRP6 mutant could be rescued from ER retention by deletion of two to four residues in the transmembrane domain. Importantly, we found that palmitoylation-deficient LRP6 was retained in the ER by a completely novel monoubiquitinationdependent ER retention mechanism. Mutation of a specific lysine indeed abolished ubiquitination of palmitoylation-deficient LRP6 and led to a rescue from ER retention. Finally, at the cell surface, we found that interplay between palmitoylation and ubiquitination was necessary for efficient Wnt signaling.he signaling of Wnt plays a critical role in a variety of developmental and adult processes in all metazoan and in diseases such as cancer (1). Canonical Wnt signaling via ␤-catenin is transduced by two receptor families: the Frizzled proteins and lipoprotein receptor-related proteins (LRPs) 5 and 6. Wnt binds to Frizzled, which then associates with LRP5/6. The cytoplasmic domains of LRP5/6, upon receptor activation by Wnt proteins, recruit the cytosolic scaffold protein axin to the membrane, displacing it from the so-called destruction complex (containing axin, glycogen synthase kinase 3␤, Wilms tumor suppressor, and ␤-catenin) involved in the degradation of ␤-catenin by the proteasome (for review see ref.2). This rescue of ␤-catenin allows it to translocate into the nucleus and interact with T cell factor (TCF)/lymphoid enhancer factor to activate transcription (3). As a result, Wnt activates multiple intracellular cascades, leading to cell differentiation, proliferation, migration, and polarity.LRP6 is a type I membrane protein with a large, 1,351-aa extracellular domain, containing multiple ␤-propeller and EGF domains, and a 220-aa-long cytoplasmic tail. As all type I transmembrane proteins, LRP6 is synthesized with an Nterminal signal sequence that targets it to the endoplasmic reticulum (ER). Folding of the LRP6 ␤-propeller domains in the lumen of the ER requires the help of an ER-resident chaperone called mesoderm development candidate 2 (Mesd) in mammals and Boca in Drosophila (4, 5). In the absence of Mesd, LRP6 is retained in the ER and thus fails to reach the cell surface.Human LRP6 contains six cysteine residues in its cytoplasmic tail, which are potential sites for palmitoylation (6). This lipid can serve to tether soluble proteins to membranes but is also found attached to the cytoplasmic domains of membrane proteins often close to the transmembrane region (7). The f...

Hydrophobic mismatch-induced clustering as a primer for protein sorting in the secretory pathway

“…The outlined scenario for facilitating the transport of mislocalized proteins by hydrophobic mismatching is consistent with very recent experimental observations on the transport behaviour of model proteins with variable transmembrane domains [13] and mutant forms of the lipoprotein receptor-related proteins LRP6 [11]. Testing the oligomeric status of such proteins in vivo, e.g.…”

“…if there is a hydrophobic mismatch, is energetically unfavourable and consequently the protein partitions (via diffusion) into a membrane domain that yields a lower mismatch. Indeed, very recent reports have further supported the central role of the hydrophobic mismatch as a key element in protein sorting [11][12][13]. In these studies the partitioning of proteins according to their TMD was either directly observed or indirectly derived from the transport behaviour of the investigated constructs.…”

Human genetic variation altering anthrax toxin sensitivity

“…A consequence of such phosphorylation is recruitment of the adapter, β-arrestin, which induces the binding of an E3 ubiquitin ligase that ubiquitinates a lysine residue in the cytosolic tail (29,30). C16 carbon acyl chains are added to one or more cytosolic cysteine residues by palmitoyltransferase (53), enabling the receptor to interact with heterotetrameric adaptor complex-dependent multimember clathrin machinery and to enter cells by clathrin-dependent endocytosis (29). Replacement of proline-357 by alanine disrupts a segment of proline repeats in the CMG2 cytoplasmic tail, possibly affecting the ability of this proline-rich region to interact with other proteins mediating the endocytic process (54).…”

The Transmembrane Domain of the Molecular Chaperone Cosmc Directs Its Localization to the Endoplasmic Reticulum

“…There is also emerging evidence that palmitoylation of Cys residues in the TMD of resident ER proteins may influence the ER retention. For example, the lipoprotein receptor-related protein 6 (LRP6) is involved in canonical Wnt signaling (67), and palmitoylation of membrane Cys residues is required for its exit from the ER; consequently mutation of Cys residues leads to ER retention (68). However, this mechanism is not likely to be involved in Cosmc retention in the ER since the Cys-19 residue in the TMD is required for disulfide-bond formation and loss of Cys leads to movement out of the ER.…”