The presentse results provide a systematic analysis of the membrane trafficking events underlying lysosomal down-regulation of the DORs. We studied focused on this particular mammalian 7TMR because its lysosomal destruction is a physiologically important regulatory process (20
), and because DORs have the the remarkable remarkable ability to down-regulate effectively via an ESCRT-dependent mechanism when their ubiquitination is prevented. Previous work identifying has described alternate (non-ESCRT) protein connectivity modulating DOR down-regulation influencing DOR trafficking after endocytosis (25
) leaves unresolved whether DORs traverse MVBs and, if so, what role. Thus we focused on defining precisely what role ubiquitination plays in mediating or controlling the the endocytic membrane trafficking of down-regulation of this mammalian 7TMR, and determining how this trafficking function affects the process of receptor proteolysis..
Our data clearly establish that internalized DORs traffic via morphologically characteristic MVBs, and localize to the endosome lumen in an ESCRT and PI3K -dependent manner, which supporting the hypothesis that DORs traverse the canonical MVB pathway represent canonical intermediates in the pathway mediating down-regulation of diverse signaling receptors (43
). Accordingly, we took several independent approaches to discern the specific functional significance of DOR ubiquitination to receptor trafficking within this pathway. First, we assessed the sorting of receptors away from bulk membrane recycling using an established flow cytometric method. Second, we investigated whether receptors traverse MVBs by using immunoelectron microscopy and using several optical imaging approaches. Third, we assessed examined the effect of intra-MVB sorting on biochemical accessibility of receptors to the endosome lumen using both pH-sensitive GFP (pHluorin) imaging and biochemical analysis of domain-specific proteolytic fragmentation.
Exclusion of DORs from bulk the rapid recycling pathway marked by TfRs was highly efficient, was was not detectably inhibited by lysine mutation of receptors, and clearly preceded the onset of proteolytic digestion of receptors measured by any of the biochemical assays. These findings define receptor sorting away from the bulk recycling route as a discrete, and early, sorting operation. Quantitative imaging revealed that uUbiquitination of DORs specifically promoted their later intralumenal localization to the intralumenal compartment of endosomes of internalized DORs. This is consistent with the ability of ubiquitination of various integral membrane proteins to promote intra-MVB sorting, of many integral membrane proteins with the two notable exceptions: First, that 1) ubiquitination of DORs was not essential for intralumenal localization, (as lysine-mutant receptors were clearly observed to access the endosome lumen both by immuno-electron microscopy and fluorescence imaging of living cells), and 2) . Second, that neither DOR ubiquitination nor intra-MVB sorting was required to prevent internalized receptors from recycling to the plasma membrane.
Together, thesethese results observations support a model with in which endocytic trafficking of the DOR to lysosomes involves two discrete molecular sorting operations that are arranged in series in the MVB pathway, and which differing in dependence on receptor ubiquitination (). Importantly, the finding that lysyl mutation of DORs reduces intralumenal sorting of DORs localization without causing any enhancement of receptor recycling rules out the alternative hypothesis that ubiquitin -independent and -dependent mechanisms function in parallel.
Model for sequential ubiquitin-independent –and dependent regulation of DOR degradation
T, and indicates tha the first operation is the the ubiquitin-dependent exclusion of internalized receptors from the rapid recycling pathway mechanism functions upstream. The first of these sequential sorting operations, effective exclusion of DORs from the bulk recycling pathway, is completely insensitive to receptor ubiquitination and is ('Sorting Step I' in ); this sorting step does not require ubiquitination of the DOR and, for this particular 7TMR, represents the major major determinant of subsequent the receptor’s ultimate endocytic trafficking fate. This is In contrast, remarkable because previous studies have shown that lysyl mutation has been shown to markedly increase s recycling of other several other signaling receptors in both yeast and mammalian cells (14
), including some the other mammalian GPCRs PAR2 and NK1R7TMRs (17
). This suggests that the upstream sorting step represents an elaboration of the down-regulation pathway that is engaged specifically by a subset of signaling receptors.
The second sorting operation in the serial sequential model is receptor ttransfer of DORs from the endosome limiting membrane to ILVs ('Sorting Step II' in ). This topological sorting operation is promoted stimulated by DOR ubiquitination, and is thus similar to corresponds to the primary sorting operation distinguishing recycling from degradative trafficking of various other signaling receptorsof 7TMRs in yeast and the EGF receptor in mammalian cells. A distinction is that, for the DOR, , relative to previously investigated signaling receptors, is that lysyl-ubiquitination promotes but is clearly not essential for topological sorting to receptor localization to ILVs. This verifies the dominant role of Sorting Step I in directing DOR trafficking to the MVB pathway, and suggests that this upstream sorting operation is sufficient to drive effective delivery of receptors to sites of ESCRT-mediated ILV formation even in the absence of receptor ubiquitination..
The Live imaging of pHluorin-tagged receptors data established indicate that intra-MVB sorting of DORs to the endosome lumen affords access of the receptor endodomain to the acidic environment of the endosome lumen. The proteolytic cleavage data analysis confirm showed, further, and extend this conclusion, a thats intra-MVB sorting selectively acceleratesd destruction of the receptor endodomain. Thus the present results can simply explain why previous studies of DOR ubiquitination have detected only a relatively subtle effect on net proteolytic destruction of DORs.
An important question for future study is how receptors devoid of any ubiquitination retain the ability to undergo sorting to undergo topological sorting to ILVs the MVB lumen. While wee note that there is considerable evidence for the existence of alternate mechanisms of intra-MVB trafficking ILV formation and cargo traffic (39
). Our , our data results suggest strongly suggest that both wild type and lysine-mutant non-ubiquitinated DORs access the endosome lumen by the canonical (ESCRT-dependent) mechanism dependent on ESCRT 0 and PI3K, rather than alternate mechanism(s) with different biochemical features (39
). The simplest hypothesis capable of explaining the present findings is that The simplest possibility is that nonon-ubiquitinated receptors, because they are unable to efficiently enter the rapid recycling pathway originating from early / sorting endosomes, are effectively 'trapped' in maturing endosomes and subsequently access the intralumenal compartment during the process of MVB biogenesis simply by lateral passive diffusion into ESCRT domains in formed at the limiting membrane. An additional, and not mutually exclusive, possibility is that alternate DOR (i.e. not mediated by ubiquitin interaction) connectivity of DORs to the ESCRT 0 (25
), as proposed here to function in Sorting Step I, persists during MVB biogenesis at later stages of endosome maturation and further to further facilitate promote ESCRT-mediated entry into the intralumenal compartment (, inset). Either or bBoth of these mechanisms are plausible based on present information, and could simply account for the ability of non-ubiquitinated DORs to undergo sorting to ILVs. Finally, wWe cannot exclude the possibilityy that topological DOR sorting of DORs trafficking to ILVss is promoted by interaction with a distinct, ubiquitinated linker protein or other receptor (and likely ubiquitinated) endocytic cargo protein, or that DORs access ILVs by lateral partitioning into biophysically distinct microdomains of the limiting membrane from which ESCRT-dependent ILV formation occurs. We note that, while that DORs DORs are known to have the ability to homo-oligomerize when present at high surface concentration, and may also and form hetero-oligomers specfically with other opioid receptor subtypes 7TMRs (45
), HEK293 cells do not express any opioid receptors endogenously. Any of these possibilities could explain the ability of DORs to undergo topological sorting in the absence of ubiquitination, and none would Further, even if DOR sorting is assisted by association with an (as yet unknown) ubiquitinated intermediary of some kind, this possibility would not fundamentally alterinvalidate the present conclusion that DORs are sorted sequentially in the MVB pathway. the present interpretation that biochemically distinct sorting steps allow mammalian cells to selectively control the ultimate trafficking fate of DORs independently from topological sorting to ILVs.
Another interesting important question for future investigation is to determine the functional significance of what functional advantage is conferred by distributing the endocytic trafficking of DORs into discrete, and sequential molecular , sorting operations in the pathway of DOR down-regulation. An An obvious attractive possibility, as mentioned above, is that thiss equential sorting could organization could provide the cell wiafford an than additional degree of freedom in the endocytic regulation of particular signaling receptors, by effectively allowing to control the cytoplasmic accessibility of internalized internalized signaling receptors to be controlled specifically and independently from independently from their their ultimate trafficking fate. There is accumulating evidence that various signaling receptors, including some 7TMRs, can signal from endosomes as well as from the plasma membrane of mammalian cells (1
). There is also evidence that intra-MVB sorting is a primary significant mechanism for terminating receptor-mediated signaling by the EGF receptoir tyrosine kinases in the endocytic pathway (2
). Thus we speculate anticipate that the ESCRT / MVB system, besides its established function in driving ubiquitin-directed destruction of various integral membrane proteins, that serves additional role(s) in controlling the duration or subcellular localization of specific receptor-mediated signaling activities. the sequential organization of biochemically discrete molecular sorting operations, established in the present study, likely provides animal cells with an additional level of specificity in regulating particular members of the largest known family of signaling receptors.