Figure 9. Model for sequential ubiquitin-independent –and dependent regulation of DOR degradation.

The wild type DOR, as well as the lysine mutant DOR0cK that cannot be ubiquitinated, undergo regulated endocytosis following ligand-induced activation (1). Receptors are prevented from traversing the default recycling pathway by 'Sorting step I', which does not require receptor ubiquitination and is sensitive to ubiquitination-independent interaction of receptors with GASPs (2). Receptors undergo topological sorting from the limiting membrane to ILVs; this represents a discrete operation that we call 'Sorting step II'. Sorting step II resembles canonical ubiquitin-dependent sorting of other cargo and requires the ESCRT. The difference is that DORs can still undergo transfer to ILVs, albeit with moderately reduced rate or efficiency, when receptor ubiquitination is prevented (3). This sequential organization of discrete sorting operations, together with (partial) ubiquitination-dependence specifically of the downstream step, explains the ability of lysine-mutant DORs to down-regulate effectively via the canonical pathway. Accordingly, lysine mutation causes a selective and partial inhibition of later proteolytic events that require protease access to the receptor’s cytoplasmic surface (4).