Differential recognition of a dileucine-based sorting signal by AP-1 and AP-3 reveals a requirement for both BLOC-1 and AP-3 in delivery of OCA2 to melanosomes

Cell types that generate unique lysosome-related organelles (LROs), such as melanosomes in melanocytes, populate nascent LROs with cargoes that are diverted from endosomes. Cargo sorting toward melanosomes correlates with binding via cytoplasmically exposed sorting signals to either heterotetrameric adaptor AP-1 or AP-3. Some cargoes bind both adaptors, but the relative contribution of each adaptor to cargo recognition and their functional interactions with other effectors during transport to melanosomes are not clear. Here we exploit targeted mutagenesis of the acidic dileucine-based sorting signal in the pigment cell-specific protein OCA2 to dissect the relative roles of AP-1 and AP-3 in transport to melanosomes. We show that binding to AP-1 or AP-3 depends on the primary sequence of the signal and not its position within the cytoplasmic domain. Mutants that preferentially bound either AP-1 or AP-3 each trafficked toward melanosomes and functionally complemented OCA2 deficiency, but AP-3 binding was necessary for steady-state melanosome localization. Unlike tyrosinase, which also engages AP-3 for optimal melanosomal delivery, both AP-1- and AP-3-favoring OCA2 variants required BLOC-1 for melanosomal transport. These data provide evidence for distinct roles of AP-1 and AP-3 in OCA2 transport to melanosomes and indicate that BLOC-1 can cooperate with either adaptor during cargo sorting to LROs.     

Differential expression of AP-2gamma and AP-2alpha during human trophoblast differentiation.

Previous studies from our laboratory demonstrated that AP-2alpha induces the expression of the hPL and hCG genes in cultured trophoblast cells. In the current study, we have shown by transient transfection studies that AP-2gamma, which is the product of a separate gene from AP-2alpha, also stimulates hPL and hCGbeta promoter activities. However, AP-2gamma mRNA levels during in vitro differentiation of human cytotrophoblast cells were strikingly different than those of AP-2alpha mRNA levels, with AP-2alpha increasing and AP-2gamma markedly decreasing during the differentiation process. The amount of AP-2gamma protein binding to AP-2 elements on the hPL promoter, as determined by supershift assays, also markedly decreased during the differentiation process. These findings strongly suggest that AP-2gamma action in cytotrophoblast cells is repressed by a co-factor(s) that inhibits AP-2gamma action or is prevented by the absence of a co-factor(s) that is essential for AP-2gamma action.     

Mitogen stimulation of T-cells increases c-Fos and c-Jun protein levels, AP-1 binding and AP-1 transcriptional activity.

We have analysed the effect of mitogenic lectins on c-Fos and c-Jun protein levels as well as on activator protein-1 (AP-1) binding and enhancer activity in Jurkat T-cells. Both c-Fos and c-Jun protein levels were increased after Con A and PHA stimulation. Since T-cell stimulation increases both intracellular Ca2+ and cAMP levels and activates protein kinase C (PKC), the possible involvement of these intracellular messengers in c-Fos and c-Jun induction was tested. PMA, which directly activates PKC, mimicked the effect of the lectins on c-Fos and c-Jun, but elevation of either intracellular Ca2+ or cAMP levels had little or no effect. The mitogen-induced increase of c-Fos and c-Jun immunoreactivity was inhibited by H-7, a kinase inhibitor with relatively high specificity for PKC, and less efficiently by H-8, a structurally related kinase inhibitor less active on PKC, but more active on cyclic nucleotide-dependent kinases. Con A stimulation was found to increase both binding of AP-1 to the AP-1 consensus sequence, TRE, and AP-1 enhancer activity, in Jurkat cells. PMA was also found to increase the AP-1 enhancer activity, whereas elevation of Ca2+ or cAMP had only minor effects. We conclude that stimulation with mitogenic lectins is sufficient to increase both c-Fos and c-Jun protein levels, AP-1 binding and AP-1 enhancer activity in Jurkat cells and that they act via mechanisms that could involve the activation of PKC.     

Tumor promotion resistant cells are deficient in AP-1 DNA binding, JunD DNA binding and JunD expression and form different AP-1-DNA complexes than promotion sensitive cells

The JB6 cell culture model is used to identify molecular determinants of susceptibility to the promotion of neoplastic transformation. Clonal variants susceptible to transformation ('P+' cells) form numerous anchorage-independent colonies in soft agar upon treatment with the phorbol ester tumor promoter TPA, whereas resistant variants ('P-' cells) do not. We now report that there is significantly less binding of activator protein-1 (AP-1) to its DNA binding site in P- cells than in P+ cells. Gel supershift assays were performed to detect association of all seven AP-1 family members with their DNA binding site in TPA-treated and -untreated P+ and P- cells. Significantly lower DNA binding and protein expression of JunD were detected in P- cells than in P+ cells. c-Jun was detected in P+, but not P-, AP-1-DNA complexes, and c-Fos was detected in P-, but not P+, AP-1-DNA complexes. These and other phenotype-specific differences in abundance and composition of AP-1-DNA complexes may play a role in the resistance of P- cells to tumor promoter-induced transformation.     

Identification of acidic dileucine signals in LRP9 that interact with both GGAs and AP-1/AP-2

The Golgi-localized, gamma-ear-containing, ADP ribosylation factor-binding family of monomeric clathrin adaptors (GGAs) is known to bind cargo molecules through short C-terminal peptide motifs conforming to the sequence DXXLL (X = any amino acid), while the heterotetrameric adaptors AP-1 and AP-2 utilize a similar but discrete sorting motif of the sequence [D,E]XXXL[L,I]. While it has been established that a single cargo molecule may contain either or both types of these acidic cluster-dileucine (AC-LL) sorting signals, there are no examples of cargo with overlapping GGA and AP-1/AP-2-binding motifs. In this study, we report that the cytosolic tail of low-density lipoprotein receptor-related protein (LRP)9 contains a bifunctional GGA and AP-1/AP-2-binding motif at its carboxy-terminus (EDEPLL). We further demonstrate that the internal EDEVLL sequence of LRP9 also binds to GGAs in addition to AP-2. Either AC-LL motif of LRP9 is functional in endocytosis. These findings represent the first study characterizing the trafficking of LRP9 and also have implications for the identification of additional GGA cargo molecules.     

Ten ERK-related proteins in three distinct classes associate with AP-1 proteins and/or AP-1 DNA.

We have identified seven ERK-related proteins ("ERPs"), including ERK2, that are stably associated in vivo with AP-1 dimers composed of diverse Jun and Fos family proteins. These complexes have kinase activity. We designate them as "class I ERPs." We originally hypothesized that these ERPs associate with DNA along with AP-1 proteins. We devised a DNA affinity chromatography-based analytical assay for DNA binding, the "nucleotide affinity preincubation specificity test recognition" (NAPSTER) assay. In this assay, class I ERPs do not associate with AP-1 DNA. However, several new "class II" ERPs do associate with DNA. p41 and p44 are ERK1/2-related ERPs that lack kinase activity and associate along with AP-1 proteins with AP-1 DNA. Class I ERPs and their associated kinase activity thus appear to bind AP-1 dimers when they are not bound to DNA and then disengage and are replaced by class II ERPs to form higher order complexes when AP-1 dimers bind DNA. p97 is a class III ERP, related to ERK3, that associates with AP-1 DNA without AP-1 proteins. With the exception of ERK2, none of the 10 ERPs appear to be known mitogen-activated protein kinase superfamily members.     

Spatial and Functional Relationship of GGAs and AP-1 in Drosophila and HeLa Cells

The GGAs [Golgi-localised, γ-ear containing, ARF (ADP ribosylation factor)-binding proteins] and the AP-1 (adaptor protein-1) complex are both adaptors for clathrin-mediated intracellular trafficking, but their relationship to each other is unclear. We have used two complementary systems, HeLa cells and Drosophila Dmel2 cells, to investigate GGA and AP-1 function. Immunoelectron microscopy of endogenous AP-1 and GGA in Dmel2 cells shows that they are predominantly associated with distinct clathrin-coated structures. Depletion of either GGA or AP-1 by RNAi does not affect the incorporation of the other adaptor into clathrin-coated vesicles (CCVs), and the cargo protein GFP-LERP (green fluorescent protein-lysosomal enzyme receptor protein) is lost from CCVs only when both adaptors are depleted. Similar results were obtained using HeLa cells treated with siRNA to deplete all three GGAs simultaneously. AP-1 was still incorporated into CCVs after GGA depletion and vice versa, and both needed to be depleted for a robust inhibition of receptor-mediated sorting of lysosomal hydrolases. In contrast, downregulation of major histocompatibility complex (MHC) class I by HIV-1 Nef, which requires AP-1, was not affected by a triple GGA knockdown. Thus, our results indicate that the two adaptors can function independently of each other.     

BLOC-1 interacts with BLOC-2 and the AP-3 complex to facilitate protein trafficking on endosomes

The adaptor protein (AP)-3 complex is a component of the cellular machinery that controls protein sorting from endosomes to lysosomes and specialized related organelles such as melanosomes. Mutations in an AP-3 subunit underlie a form of Hermansky-Pudlak syndrome (HPS), a disorder characterized by abnormalities in lysosome-related organelles. HPS in humans can also be caused by mutations in genes encoding subunits of three complexes of unclear function, named biogenesis of lysosome-related organelles complex (BLOC)-1, -2, and -3. Here, we report that BLOC-1 interacts physically and functionally with AP-3 to facilitate the trafficking of a known AP-3 cargo, CD63, and of tyrosinase-related protein 1 (Tyrp1), a melanosomal membrane protein previously thought to traffic only independently of AP-3. BLOC-1 also interacts with BLOC-2 to facilitate Tyrp1 trafficking by a mechanism apparently independent of AP-3 function. Both BLOC-1 and -2 localize mainly to early endosome-associated tubules as determined by immunoelectron microscopy. These findings support the idea that BLOC-1 and -2 represent hitherto unknown components of the endosomal protein trafficking machinery.     

Differential requirements for AP-2 in clathrin-mediated endocytosis

AP-2 complexes are key components in clathrin-mediated endocytosis (CME). They trigger clathrin assembly, interact directly with cargo molecules, and recruit a number of endocytic accessory factors. Adaptor-associated kinase (AAK1), an AP-2 binding partner, modulates AP-2 function by phosphorylating its mu2 subunit. Here, we examined the effects of adenoviral-mediated overexpression of WT AAK1, kinase-dead, and truncation mutants in HeLa cells, and show that AAK1 also regulates AP-2 function in vivo. WT AAK1 overexpression selectively blocks transferrin (Tfn) receptor and LRP endocytosis. Inhibition was kinase independent, but required the full-length AAK1 as truncation mutants were not inhibitory. Although changes in mu2 phosphorylation were not detected, AAK1 overexpression significantly decreased the phosphorylation of large adaptin subunits and the normally punctate AP-2 distribution was dispersed, suggesting that AAK1 overexpression inhibited Tfn endocytosis by functionally sequestering AP-2. Surprisingly, clathrin distribution and EGF uptake were unaffected by AAK1 overexpression. Thus, AP-2 may not be stoichiometrically required for coat assembly, and may have a more cargo-selective function in CME than previously thought.     

Recognition sites for clathrin-associated proteins AP-2 and AP-3 on clathrin triskelia.

AP-2 and AP-3 are cellular proteins that drive the in vitro polymerization of clathrin triskelia into cage structures. The interaction of these two types of assembly proteins (APs) with preassembled clathrin cages has been studied in order to identify the sites on the triskelia required for binding. Comparing binding of the APs to intact or to proteolytically clipped cages, we attempted to distinguish between binding to the terminal domain, the globular end of the heavy chain, and binding to the hub of the clathrin triskelia, the portion that remains assembled after trypsin treatment. AP-3 binds to intact clathrin cages but not to those that were treated with trypsin. AP-3 also bound to cages consisting solely of clathrin heavy chains; proteolysis of these cages also eliminated AP-3 binding. In addition, AP-3 did not bind to either isolated hubs or terminal domains that had been immobilized on Sepharose. These data indicate that clathrin light chains are not required for binding of AP-3, and that neither terminal domain nor hubs alone will suffice. However, an intact heavy chain is both necessary and sufficient for the binding of AP-3. Previous work has demonstrated one binding site for AP-2 on proteolyzed cages containing only clathrin hubs; the existence of a second binding site associated with the terminal domain was hypothesized. Here we provide direct evidence for recognition by AP-2 of isolated terminal domains immobilized on Sepharose and show that the core of the AP-2 molecule is responsible for this interaction. These results provide the first demonstration of a functional role for the conserved terminal domain of the clathrin heavy chain.