Binding of agonist to surface receptors initiates kinase cascades, modulating signaling pathways and gene expression within the target cell. However, ligand can also alter trafficking of the receptor, in ways specific to the ligand, receptor isoform, and cell type. For many membrane receptors, ligand binding is followed by increased rates of internalization and processing. The pathways and subsequent events have immediate consequences for signal transduction (for reviews, see Refs. 47
). Our system, permitting study of endogenously expressed receptors rather than transiently transfected receptors, avoids complications that may be secondary to overexpression, such as a high proportion of improperly folded or mistrafficked receptors (for review, see Ref. 52
). Here we demonstrated that PRL dramatically down-regulates the endogenous lPRLR in these breast cancer cells. This fall in lPRLR levels was temporally linked to the appearance of a cell-associated ECD-containing receptor fragment, which is dependent on proteasomal, but not lysosomal or metalloprotease, activities. These findings demonstrated complex effects of ligand on lPRLR trafficking and pointed to a novel role for proteasomes in limited proteolysis resulting in generation of a stable lPRLR cleavage product.
Although a role for lysosomes in degradation of the PRLR has been established (for review, see Ref. 10
), the current studies indicate a role for proteasomes in the post-ligand binding fate of the lPRLR as well. Our data were compatible with the emerging story of many receptor tyrosine kinases and G-protein coupled receptors, which employ ubiquitination to direct trafficking in response to ligand, terminating in degradation in lysosomes (for reviews, see Refs. 38
). Many details remain to be worked out, including the identity of the ubiquitinated moiety(s), the nature and location of the ubiquitin chains, the PRL-initiated signal(s) leading to this modification, and the relationship between this event(s) and lysosomal degradation. SCFβ-TrCP
has been implicated in ubiquitination of the lPRLR (11
). Whether this is the only E3 ubiquitin ligase involved in ubiquitin-mediated trafficking of the lPRLR remains to be determined. Other E3 ubiquitin ligases have been implicated in ubiquitination of cytokine receptors, including p33RUL
for the erythropoietin receptor (58
), and several, including c-Cbl and Nedd4 family members, mediate trafficking of the epidermal growth factor receptor by direct ubiquitination of the receptor or associated proteins (for reviews, see Refs. 57
). Unlike the closely related GHR and another cytokine receptor, the interferon-α receptor, the lPRLR does not require the ubiquitin-conjugating system for internalization (37
). Our current studies are consistent with our previous report of the requirement for multiple motifs within the cytoplasmic domain of the lPRLR for optimal internalization, as opposed to the single the Ub-dependent endocytosis motif of the GHR (7
). Thus the PRLR appears to more closely resemble the erythropoietin receptor (61
) and interleukin-2 receptor β (41
), other members of the cytokine receptor family, in this regard.
Our studies also revealed that PRL-initiated proteasomal activity is critical for generation of a cell-associated 50-kDa PRLR fragment containing the ECD. The size of this fragment suggested that it results from a cleavage event(s) in the vicinity of the transmembrane domain. Although this PRLR-ECD fragment suggested a “sheddase,” such as that reported for multiple other transmembrane receptors, including the cytokine receptors, GHR (12
), the interleukin-6 receptor (64
), ErbB4 (13
), and Notch (65
), the apparent regulation of this lPRLR proteolytic event is quite different. Cleavage of the lPRLR was insensitive to protein kinase C agonists and inhibitors, as well as inhibition of the metalloproteases implicated in secretion of these other receptor fragments. Interestingly, the etiology of this cell-associated PRLR-ECD fragment is strikingly similar to that reported for some membrane bound transcription factors (for reviews, see Refs. 42
). Proteasomal cleavage of these transmembrane proteins occurs in polysomes or the endoplasmic reticulum. However, the origination of this lPRLR fragment from surface receptor, and independence from concurrent translation, were not consistent with these locations. Our data suggested that the lPRLR is cleaved after ligand-stimulated internalization, which would be a novel site for this limited proteasomal action. A proteasome-dependent fragment from the common β (βc) subunit of hematopoietic cytokine receptors displays intriguing similarities, although few details are known, suggesting that the PRLR may not be alone in this respect (67
). Clearly, the identity and function of this PRLR fragment require further study. Whether it is merely an intermediate in degradation of the PRLR or transmits additional signals, such as that proposed for other mitogenic receptor fragments (for reviews, see Refs. 13
), is under investigation. Although early reports offered tantalizing suggestions of PRLR translocation into the nucleus (68
), this has remained controversial (69
). Our data suggested that this possibility should be reevaluated.
The mechanism whereby PRL initiates PRLR cleavage is currently under study. Inhibition of multiple kinase cascades did not block formation of the PRLR-ECD fragment.5
Whether PRL induces a conformational change in the lPRLR, permitting cleavage, or initiates this process by signaling pathways not examined remains to be determined. Little is known about PRL-initiated signals that may result in serine/threonine phosphorylation of the lPRLR and/or associated proteins. These modifications mediate many interactions important for post-ligand trafficking and facilitate ubiquitination of many membrane receptors (56
). Indeed, phosphorylation of Ser-349 was required for PRLR recognition by SCFβ-TrCP in vitro
In conclusion, we have provided evidence that proteasomes mediate ligand-stimulated generation of an ECD-containing fragment and lPRLR degradation in breast cancer cells. Differences in PRL-activated signaling pathways (3
) and endocytic motifs among the alternatively spliced PRLR isoforms (7
) suggest that post-ligand trafficking of the short isoforms will be distinct from the lPRLR. Understanding these processes, their regulation, and their interrelationships with signaling cascades in different target cells will increase our knowledge of PRL actions in physiologic activities and provide the basis to modulate its actions in pathological conditions, including breast cancer.