As previous experiments have focused on short-term characterization of Mer activation and functional effects associated with Mer expression, this paper for the first time examines the direct consequences of long-term Gas6 exposure on Mer. We used an in vitro
model of prolonged Gas6 exposure to study Mer under conditions that more closely resemble the Gas6-replete environment that exists in the bone marrow 
and plasma 
—one in which the constant interaction between Gas6 and Mer presumably sustains downstream signaling activity and promotes leukemic cell survival in vivo
. These studies provide insight into several novel aspects of Mer that redefine its function beyond the role of a signal transducer.
In human leukemia cell lines, continuous Gas6 exposure promoted expression of a partially N-glycosylated form of Mer, a glycoform that developed from de novo partial glycosylation of a newly synthesized protein. Collectively, the data from the glycan profiling and mechanistic studies suggest that persistent ligand exposure induces a switch to preferentially express the same partial Mer glycoform that normally exists as a minor form in the absence of Gas6. This idea is supported by the similarities in molecular weight and EndoH susceptibility (), which suggest a shared glycan profile; additionally, the comparable response of the partial glycoforms to tunicamycin () indicates that they both utilize the same mechanism of post-translational modification, lending further support to this idea from a mechanistic perspective. The data obtained from tunicamycin-mediated glycosylation inhibition also provide novel insight into the receptor dynamics of Mer: by distinguishing newly synthesized (and non-glycosylated) proteins from mature receptors, we demonstrate that translation of new Mer occurs within a few hours. Based on the minimal amount of fully glycosylated Mer remaining after 24 hours of tunicamycin exposure, it is also evident that the majority of pre-existing receptor is recycled within this time frame. Although Mer dynamics still remain largely uncharacterized, this finding has important implications for drug design, as inhibitors with shorter half-lives would not likely have a sustained effect on Mer activity.
While Gas6-favored expression of the partial glycoform was associated with diminished levels of Mer on the cell surface (), exposure to BFA or TM, which restrict or completely inhibit glycosylation, respectively, demonstrated that the reduction in surface Mer was likely due to its partially glycosylated state rather than a direct consequence of ligand exposure (). We also now report expression of Mer in the nucleus, illustrated both by immunofluorescent imaging and subcellular fractionation experiments ( and ). That Mer is expressed in any of the nuclear compartments—and as an intact, rather than cleaved, receptor—is a novel finding for this protein, as well as one that has not yet been reported for either of the other TAM receptors. However, several other studies have established the presence of full-length RTKs in the nucleus 
, including members of the epidermal growth factor receptor (EGFR) family of proteins 
, fibroblast growth factor receptor 
, insulin receptor 
, and insulin-like growth factor-1 receptor 
. Expression of c-Met 
and RON 
, receptors closely related to the TAM family 
, has also been demonstrated in the nucleus.
Although the conserved nature of the NLS and NES () suggest that Axl and Tyro3 may also localize to the nucleus, our efforts presently aim to determine the mechanistic bases of Mer translocation to the nucleus; as nuclear RTKs are associated with various changes in biologic activity 
, we are also focused on elucidating the functional significance of nuclear Mer expression. Based on reports of nuclear RTK-related effects on gene expression—including regulation by direct transactivation as well as through complexed interactions 
—ChIP-Seq profiling experiments are currently underway to identify regions of DNA influenced by Mer, which is present within the chromatin-bound fraction ( and ).
Several studies have reported differential glycosylation of Mer 
but none have explored the functional effects associated with this modification. The contrasting patterns of glycoform expression observed within each nuclear fraction () suggest that the nuclear compartments may exhibit distinct preferences for specific Mer glycoforms. This glycoform-influenced nuclear sublocalization indicates functional consequences of Mer glycosylation, suggesting that particular N-glycan modifications on Mer may influence its affinity for DNA and/or partner proteins within the nucleus. Our data also highlight that Mer does not exclusively occupy the subcellular locales predicted by its glycosylation profile, suggesting that such modifications do not always restrict or define function. This idea is supported by a previous study emphasizing how the functional maturity of a protein is not necessarily defined by its degree of glycosylation: Krysov et al. found that despite the mannosylated, so-called “immature” nature of the IgM μ chain expressed in chronic lymphocytic leukemia samples, increased expression of this partial glycoform—which could be induced by persistent antigen exposure—retained the same functional capabilities as its fully glycosylated, “mature” counterpart 
Our observations widen the scope of potential Mer function and supplement its once-singular role as a surface receptor. Both the presence of Mer in the nucleus and the kinase-independent formation of the partial glycoform suggest that the oncogenic effects of Mer may, at least in part, be mediated through mechanisms involving its nuclear localization and not exclusively through increased activation at the cell surface. While previous studies have established Mer as an attractive therapeutic target in leukemia, the current focus on developing Mer-specific inhibitors emphasizes the importance of characterizing Mer function as thoroughly as possible, as the ability to effectively target Mer is limited by our understanding of its functions. The novel finding of Mer expression in the nucleus—and especially the presence of nuclear localization and export sequences—broadens the realm of potential approaches to target Mer for therapeutic purposes.