Skeletal muscle and adipocytes are specifically able to elaborate an intracellular pool of GLUT4 glucose transporters that move to the cell surface in response to insulin and thus modulate blood glucose homeostasis. Considerable morphological and biochemical evidence supports a model for GLUT4 trafficking, whereby approximately half the intracellular GLUT4 is targeted to a specific storage vesicle pool called GSVs or IRVs that is relatively deficient in other endosomal trafficking markers such as the transferrin receptor (TfR) and undergoes a marked increase in its rate of exocytosis in response to insulin (Simpson et al., 2001
; Bryant et al., 2002
). The molecular bases for this increased exocytic rate, the formation of GSVs, and the segregation of GLUT4 from endosomal markers such as the TfR remain incompletely understood.
To address this issue, we and others have used purification of GLUT4-rich vesicles to identify their protein content by immunological and biochemical (proteomics) means. These protocols have resulted in the identification of numerous proteins such as VAMP2 (Cain et al., 1992
) and sorting receptors such as M6PR (Kandror and Pilch, 1996
) and sortillin (Lin et al., 1997
; Morris et al., 1998
) that one might expect to be involved in vesicular traffic. Importantly and as cited in the introduction, IRAP was also identified in this way as marker for GSV trafficking that is apparently more abundant than GLUT4 itself. Indeed, we have shown that IRAP is directed to GSVs during the course of 3T3-L1 adipocyte differentiation before significant GLUT4 expression occurs (El-Jack et al., 1999
). Moreover, in engineered adipocytes, IRAP can become essentially the only major cargo constituent of GSVs and it responds to insulin in a manner similar or identical to GSVs from 3T3-L1 cells (Gross et al., 2004
). Thus, it can be expected that IRAP must interact with additional cellular proteins, adaptors, and tethers, to undergo proper targeting and trafficking. In particular the cytoplasmic sequences of IRAP likely interact with cytosolic proteins for steps in vesicle formation and targeting.
In this study, we used chitin binding domain (CBD) protein fused to the cytosolic sequence of IRAP, residues 1–109, to identify possible IRAP-binding proteins that might function in GSV trafficking. This fusion protein has the same orientation as does full-length IRAP in vesicles, and thus, we used it to identify p115 as an IRAP-interacting protein by a variety of criteria. Immobilized CBD-IRAP specifically binds p115 from the cytosol of 3T3-L1 cells () and rat adipocytes (not shown). Anti-IRAP antibody coimmunoprecipitates p115 from 3T3-L1 adipocytes (), and pulldown assays demonstrate that CBD-IRAP binds to the N-terminus of p115 as well as full-length protein (). Finally, immunofluorescence shows partial colocalization of p115 with GLUT4 () and IRAP (unpublished data). Importantly, expression of the N-terminal, membrane-binding sequence of p115 completely disrupts insulin-dependent GLUT4 and IRAP translocation () with no effect on GLUT1 or the Golgi marker GM130 (Figures and ). Thus we conclude that p115 plays a specific role in insulin-dependent GSV trafficking.
The question remains as to where in the cell this IRAP-p115 interaction occurs and if there is any insulin-dependent regulation of this interaction. With regard to the latter issue, phosphorylation of p115 on serine 942 by casein kinase (Brunati et al., 2001
) has been reported to enhance its binding to GM130 on Golgi membranes (Brandon et al., 2003
), but there are no reports that insulin or other hormones can effect the phosphorylation of p115. In our experiments, we do not see a significant insulin-dependent mobility shift of bulk p115 () that might reflect this phosphorylation, but insulin does seem to effect the p115-IRAP interaction and decrease the efficiency of the IRAP coimmunoprecipitation, while simultaneously increasing the amount of p115 pulled down (). Efforts to further understand this phenomenon are underway. The development of GSVs occurs during the differentiation of adipocytes, presumably as a result of the expression of an as yet unknown protein or proteins that initially results in the sequestration of IRAP, then GLUT4 (El-Jack et al., 1999
). It is unlikely that p115 is one of these putative proteins as its expression is unchanged over the 8–10 d required for adipocytes to fully differentiate (unpublished data)
As for the cellular locus of this interaction, p115 has been described to play a variety of roles in vesicular transport at different cellular loci. In vitro vesicle fusion assays suggest p115 to be a general factor required for transport vesicle fusion to target membranes (Barroso et al., 1995
; Sapperstein et al., 1995
), in the present example, presumably the plasma membrane. And although the intensity of p115 fluorescence shows it to be mainly perinuclear and similar to syntaxin 6 and GM130 in adipocytes (), subcellular fractionation reveals it to be ubiquitous in adipocytes membrane compartments, including the PM, as well as in the cytosol (). As additional support for a possible functional role for p115 localization at the plasma membrane, we recently detected this protein in detergent-resistant PM lipid rafts isolated from insulin-treated adipocytes (unpublished data). We did not observe a significant signal for p115 in Western blots of immunoadsorbed GLUT4 vesicles (not shown).
A possible site of p115 function relevant to GSV trafficking is the trans-Golgi apparatus, which has been suggested to play a part in the trafficking of GSVs/GLUT4 based, in part, on colocalization of GLUT4 with the trans-Golgi marker, syntaxin6 (Shewan et al., 2003
). However, whereas the expression of the p115-N-terminus disperses the GLUT4 signal throughout the cytosol and blocks its translocation, this construct is without effect on GM130 distribution (). Moreover, p115 function has usually been localized to the cis
and medial Golgi where it attaches COP1-coated transport vesicles to the Golgi membrane and assists in vesicle fusion by assembling SNARE pin complexes (Shorter et al., 2002
). However, p115 in adipocytes is clearly distributed throughout the cell ().
How does the N-terminal domain of p115 disrupt GLUT4 distribution and translocation? We postulate that overexpressing the membrane/vesicle-binding domain of p115 allows it to displace endogenous full-length protein and release/disperse the GSVs so that they are no longer in position to interact with other cellular proteins necessary for their translocation and proper trafficking. Such proteins could be the actin filament–interacting protein, FHOS, which was cloned recently in our lab by the yeast two-hybrid system using IRAP as bait (Tojo et al., 2003
). Other putative GSV-binding proteins include Tankyrase, a Golgi-associated MAPK substrate, which was also shown to interact with the cytoplasmic domain of IRAP by yeast two-hybrid (Chi and Lodish, 2000
) and TUG (tether containing UBX domain for GLUT4), which was shown to bind GSVs by a translocation assay (Bogan et al., 2003
). It remains to be determined at what step these proteins act in insulin-regulated GSV trafficking.
What is the role of GLUT4 cytoplasmic sequences in GSV trafficking? IRAP null mice exhibit apparently normal GLUT4 translocation (Keller et al., 2002
), indicating, as expected, that the cytoplasmic domains of GLUT4 possess the information requisite for proper GSV trafficking. Indeed and as noted above, dileucine and acidic cluster sequences in IRAP's N-terminus are very similar to those in the GLUT4 C-terminus. We were unable to show direct p115-GLUT4 interactions with C-terminus-GST fusion proteins (unpublished data) either because the orientation of the fusion protein was such to prevent this interaction, or because GLUT4 has putative trafficking information in three noncontiguous cytoplasmic domains, all of which might be required for interaction with p115 (Khan et al., 2004
). Still, it seems highly likely that the cytosolic sequences of both IRAP and GLUT4 play important roles in the cellular itinerary of GSVs. Finally, it also seems quite likely that p115 is not the only cytoplasmic protein that interacts with GSV components and we are continuing our efforts to identify more such molecules.