Lumenal and Transmembrane Domains Play a Role in Sorting Type I Membrane Proteins on Endocytic Pathways

Previous studies have shown that when the cytosolic domains of the type I membrane proteins TGN38 and lysosomal glycoprotein 120 (lgp120) are added to a variety of reporter molecules, the resultant chimeric molecules are localized to the trans-Golgi network (TGN) and to lysosomes, respectively. In the present study we expressed chimeric constructs of rat TGN38 and rat lgp120 in HeLa cells. We found that targeting information in the cytosolic domain of TGN38 could be overridden by the presence of the lumenal and transmembrane domains of lgp120. In contrast, the presence of the transmembrane and cytosolic domains of TGN38 was sufficient to deliver the lumenal domain of lgp120 to the trans-Golgi network. On the basis of steady-state localization of the various chimeras and antibody uptake experiments, we propose that there is a hierarchy of targeting information in each molecule contributing to sorting within the endocytic pathway. The lumenal and cytosolic domains of lgp120 contribute to sorting and delivery to lysosomes, whereas the transmembrane and cytosolic domains of TGN38 contribute to sorting and delivery to the trans-Golgi network.     

A di-leucine motif mediates endocytosis and basolateral sorting of macrophage IgG Fc receptors in MDCK cells.

An important function of the low affinity IgG Fc receptor FcRII-B2 (FcR) on macrophages is the internalization of soluble antigen-antibody complexes for lysosomal degradation. Most endocytic receptors possess tyrosine-containing cytoplasmic determinants required for endocytosis. In many proteins, signals which overlap with the endocytosis determinant and share the same critical tyrosine residue also mediate basolateral sorting in the trans-Golgi network of epithelial cells. Despite the presence of two tyrosine residues in the FcR cytosolic domain, neither one is absolutely required for coated pit localization or basolateral targeting. Nevertheless, a short domain of 13 residues containing one of the non-critical tyrosine residues mediates endocytosis and basolateral delivery. Alanine scan mutagenesis of this region now revealed a critical role of a leucine-leucine motif in both events. These findings suggest that endocytosis and basolateral sorting can be mediated by both tyrosine- and di-leucine-based signals and confirm the close relationship between the two determinants already observed for 'classical' tyrosine-dependent motifs.     

Polarized sorting of the polymeric immunoglobulin receptor in the exocytotic and endocytotic pathways is controlled by the same amino acids.

Polarized epithelial cells can sort plasma membrane proteins to the apical or basolateral domain either by direct targeting from the trans-Golgi network (TGN) or by targeting to one surface, followed by endocytosis and transcytosis to the opposite surface. In Madin-Darby canine kidney (MDCK) cells, targeting of the polymeric immunoglobulin receptor (pIgR) to the basolateral surface is controlled by a sorting signal residing in the membrane proximal 17 amino acids of the cytoplasmic domain of this receptor. We have recently found that individual mutations at any of three residues in this signal, His656, Arg657 and Val660, substantially decrease targeting from the TGN to the basolateral surface and correspondingly increase targeting from the TGN to the apical surface. Here we report that these mutations decrease the recycling of basolaterally endocytosed pIgR to that surface, and correspondingly increase its transcytosis to the apical surface. This effect occurred in mutant pIgRs that either contained the full-length cytoplasmic domain or were truncated to contain only the 17-residue basolateral targeting signal, and was independent of phosphorylation of pIgR at Ser664. Our results indicate that polarized sorting of the pIgR in the endocytotic and exocytotic pathways are controlled by the same amino acids.     

Differential use of two AP-3-mediated pathways by lysosomal membrane proteins

The adaptor protein complex AP-3 is involved in the sorting of lysosomal membrane proteins to late endosomes/lysosomes. It is unclear whether AP-3-containing vesicles form at the trans-Golgi network (TGN) or early endosomes. We have compared the trafficking routes of endolyn/CD164 and 'typical' lysosomal membrane glycoproteins (lgp120/lamp-1 and CD63/lamp-3) containing cytosolic YXXPhi-targeting motifs preceded by asparagine and glycine, respectively. Endolyn, which has a NYHTL-motif, is concentrated in lysosomes, but also occurs in endosomes and at the cell surface. We observed predominant interaction of the NYHTL-motif with the mu-subunits of AP-3 in the yeast two-hybrid system. Endolyn was mislocalized to the cell surface in AP-3-deficient pearl cells, confirming a major role of AP-3 in endolyn traffic. However, lysosomal delivery of endolyn (or a NYHTL-reporter), but not GYXXPhi-containing proteins, was practically abolished when AP-2-mediated endocytosis or traffic from early to late endosomes was inhibited in NRK and 3T3 cells. This indicates that endolyn is mostly transported along the indirect lysosomal pathway (via the cell surface), rather than directly from the TGN to late endosomes/lysosomes. Our results suggest that AP-3 mediates lysosomal sorting of some membrane proteins in early endosomes in addition to sorting of proteins with intrinsically strong AP-3-interacting lysosomal targeting motifs at the TGN.     

Sorting competition with membrane-permeable peptides in intact epithelial cells revealed discrimination of transmembrane proteins not only at the trans-Golgi network but also at pre-Golgi stages

Transmembrane proteins destined to the basolateral cell surface of epithelial cells contain in their cytosolic domain at least two classes of sorting signals: one class promotes exit from the endoplasmic reticulum (ER) and transport to the Golgi complex, and the other class operates at the trans-Golgi network (TGN) specifying segregation into basolateral exocytic pathways. Both kinds of addressing motifs are quite diverse among different proteins. It is unclear to what extent this feature reflects alternative decoding mechanisms or variations in motifs recognized by the same sorting factor. Here we applied a novel strategy based on permeable peptide technology and temperature-sensitive model proteins to study competition between cytosolic sorting motifs in the context of mammalian living cells. We used the transduction domain of HIV-1 Tat protein to make a membrane-permeable peptide of the cytosolic tail of GtsO45, which contains a well characterized ER exit di-acidic (DIE) motif and a tyrosine-based basolateral sorting signal (YTDI). This peptide added to the media inhibited transport of GtsO45 from both ER-to-Golgi and TGN-to-basolateral cell surface in transfected Madin-Darby canine kidney cells. Instead, it did not affect the exocytic trafficking of a GtsO45-derived chimeric protein bearing 30 juxtamembrane residues from the cytosolic domain of the epidermal growth factor receptor that contains a variant ER exit motif (ERE) and an unconventional proline-based basolateral sorting signal. These results not only proved the feasibility of competing for sorting events in intact cells but also showed that distinct plasma membrane proteins can be discriminated at pre-TGN stages, and that basolateral sorting involves different recognition elements for tyrosine-based motifs and an unconventional basolateral motif.     

The tyrosine-based lysosomal targeting signal in lamp-1 mediates sorting into Golgi-derived clathrin-coated vesicles.

Diversion of membrane proteins from the trans-Golgi network (TGN) or the plasma membrane into the endosomal system occurs via clathrin-coated vesicles (CCVs). These sorting events may require the interaction of cytosolic domain signals with clathrin adaptor proteins (APs) at the TGN (AP-1) or the plasma membrane (AP-2). While tyrosine- and di-leucine-based signals in several proteins mediate endocytosis via cell surface CCVs, segregation into Golgi-derived CCVs has so far only been documented for the mannose 6-phosphate receptors, where it is thought to require a casein kinase II phosphorylation site adjacent to a di-leucine motif. Although recently tyrosine-based signals have also been shown to interact with the mu chain of AP-1 in vitro, it is not clear if these signals also bind intact AP-1 adaptors, nor if they can mediate sorting of proteins into AP-1 CCVs. Here we show that the cytosolic domain of the lysosomal membrane glycoprotein lamp-1 binds AP-1 and AP-2. Furthermore, lamp-1 is present in AP-1-positive vesicles and tubules in the trans-region on the Golgi complex. AP-1 binding as well as localization to AP-1 CCVs require the presence of the functional tyrosine-based lysosomal targeting signal of lamp-1. These results indicate that lamp-1 can exit the TGN in CCVs and that tyrosine signals can mediate these sorting events.     

Recycling of furin from the plasma membrane. Functional importance of the cytoplasmic tail sorting signals and interaction with the AP-2 adaptor medium chain subunit

The predominant intracellular localization of the eukaryotic subtilisin-like endoprotease furin is the trans-Golgi network (TGN), but a small fraction is also found on the cell surface. Furin on the cell surface is internalized and delivered to the TGN. The identification of three endocytosis motifs, a tyrosine (YKGL(765)) motif, a leucine-isoleucine (LI(760)) motif, and a phenylalanine (Phe(790)) signal, in the furin cytoplasmic domain suggested that endocytosis of furin occurs via an AP-2/clathrin-dependent pathway. Since little is known about proteins containing multiple sorting components in their cytoplasmic domain, the combination of diverse internalization signals in the furin tail raised the question of their individual role. Here we present data showing that the furin tail interacts with the medium (micro2) subunit of the AP-2 plasma membrane-specific adaptor complex in vitro and that this interaction primarily depends on recognition of the tyrosine-based sorting signal and to less extent on the leucine-isoleucine motif. We further provide evidence that the three endocytosis signals are of different functional importance for furin internalization and retrieval to the TGN in vivo, with the tyrosine-based motif being the major determinant, followed by the phenylalanine signal, whereas the leucine-isoleucine motif is only a minor component. Finally, we report that phosphorylation of the furin tail by casein kinase II is not only important for efficient interaction with micro2 and internalization from the plasma membrane but also determines fast retrieval of the protein from the plasma membrane to the TGN.     

Stem cell factor presentation to c-Kit. Identification of a basolateral targeting domain

Stem cell factor (also known as mast cell growth factor and kit-ligand) is a transmembrane growth factor with a highly conserved cytoplasmic domain. Basolateral membrane expression in epithelia and persistent cell surface exposure of stem cell factor are required for complete biological activity in pigmentation, fertility, learning, and hematopoiesis. Here we show by site-directed mutagenesis that the cytoplasmic domain of stem cell factor contains a monomeric leucine-dependent basolateral targeting signal. N-terminal to this motif, a cluster of acidic amino acids serves to increase the efficiency of basolateral sorting mediated by the leucine residue. Hence, basolateral targeting of stem cell factor requires a mono-leucine determinant assisted by a cluster of acidic amino acids. This mono-leucine determinant is functionally conserved in colony-stimulating factor-1, a transmembrane growth factor related to stem cell factor. Furthermore, this leucine motif is not capable of inducing endocytosis, allowing for persistent cell surface expression of stem cell factor. In contrast, the mutated cytoplasmic tail found in the stem cell factor mutant Mgf(Sl17H) induces constitutive endocytosis by a motif that is related to signals for endocytosis and lysosomal targeting. Our findings therefore present mono-leucines as a novel type of protein sorting motif for transmembrane growth factors.     

Transport of the lysosomal membrane glycoprotein lgp120 (lgp-A) to lysosomes does not require appearance on the plasma membrane

We have used stably transfected CHO cell lines to characterize the pathway of intracellular transport of the lgp120 (lgp-A) to lysosomes. Using several surface labeling and internalization assays, our results suggest that lgp120 can reach its final destination with or without prior appearance on the plasma membrane. The extent to which lgp120 was transported via the cell surface was determined by two factors: expression level and the presence of a conserved glycine-tyrosine motif in the cytoplasmic tail. In cells expressing low levels of wild-type lgp120, the majority of newly synthesized molecules reached lysosomes without becoming accessible to antibody or biotinylation reagents added extracellularly at 4 degrees C. With increased expression levels, however, an increased fraction of transfected lgp120, as well as some endogenous lgp-B, appeared on the plasma membrane. The fraction of newly synthesized lgp120 reaching the cell surface was also increased by mutations affecting the cytoplasmic domain tyrosine or glycine residues. A substantial fraction of both mutants reached the surface even at low expression levels. However, only the lgp120G----A7 mutant was rapidly internalized and delivered from the plasma membrane to lysosomes. Taken together, our results show that the majority of newly synthesized wild-type lgp120 does not appear to pass through the cell surface en route to lysosomes. Instead, it is likely that lysosomal targeting involves a saturable intracellular sorting site whose affinity for lgp's is dependent on a glycine-tyrosine motif in the lgp120 cytoplasmic tail.     

Multiple endocytic trafficking pathways of MHC class I molecules induced by a Herpesvirus protein

The K3 protein of a human tumor-inducing herpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV), down-regulates major histocompatibility complex (MHC) class I surface expression by increasing the rate of endocytosis. In this report, we demonstrate that the internalization of MHC class I by the K3 protein is the result of multiple, consecutive trafficking pathways that accelerate the endocytosis of class I molecules, redirect them to the trans-Golgi network (TGN), and target MHC class I to the lysosomal compartment. Remarkably, these actions of K3 are functionally and genetically separable; the N-terminal zinc finger motif and the central sorting motif are involved in triggering internalization of MHC class I molecules and redirecting them to the TGN. Subsequently, the C-terminal diacidic cluster region of K3 is engaged in targeting MHC class I molecules to the lysosomal compartment. These results demonstrate a novel trafficking mechanism of MHC class I molecules induced by KSHV K3, which ensures viral escape from host immune effector recognition.     

The cytoplasmic tail of lysosomal acid phosphatase contains overlapping but distinct signals for basolateral sorting and rapid internalization in polarized MDCK cells.

Lysosomal acid phosphatase (LAP) is synthesized as a type I membrane glycoprotein and targeted to lysosomes via the plasma membrane. Its cytoplasmic tail harbours a tyrosine-containing signal for rapid internalization. Expression in Madine-Darby canine kidney cells results in direct sorting to the basolateral cell surface, rapid endocytosis and delivery to lysosomes. In contrast, a deletion mutant lacking the cytoplasmic tail is delivered to the apical plasma membrane where it accumulates before it is slowly internalized. A chimeric protein, in which the cytoplasmic tail of LAP is fused to the extracytoplasmic and transmembrane domain of the apically sorted haemagglutinin, is sorted to the basolateral plasma membrane. A series of truncation and substitution mutants in the cytoplasmic tail was constructed and comparison of their polarized sorting and internalization revealed that the determinants for basolateral sorting and rapid internalization reside in the same segment of the cytoplasmic tail. The cytoplasmic factors decoding these signals, however, tolerate distinct mutations indicating that different receptors are involved in sorting at the trans-Golgi network and at the plasma membrane.     

In vitro binding study of adaptor protein complex (AP-1) to lysosomal targeting motif (LI-motif)

Lysosomal membrane glycoproteins carry targeting information in cytoplasmic regions. Two distinct targeting motifs in these regions, GY (glycine-tyrosine) and LI (leucine-isoleucine), have been identified and characterized. Accumulating evidence suggests that the adaptor complexes (AP-1, AP-2, and AP-3) recognize this information in cytoplasmic tails of transmembrane proteins. Here we report two different in vitro analyses (affinity beads and surface plasmon resonance) which revealed specific interaction between the cytoplasmic tail of LGP85 and AP-1 but not so with AP-2. We also noted requirement of the LI motif of the LGP85 tail in binding to the AP-1 complex. Our data and others which indicated the binding of AP-3 to the LIMP II (synonym of LGP85) tail suggest that the cytoplasmic tail of LGP85 interacts with AP-1 at the trans-Golgi network (TGN) and AP-3 at late endosomes, respectively. We propose that this sequential interaction between the lysosomal targeting signal and distinct APs along its transport pathway is responsible for the critical sorting of lysosomal membrane proteins and/or the potential proofreading system of mistargeted molecules.     

Regulation of protein traffic in polarized epithelial cells

The plasma membrane of polarized epithelial cells is divided into apical and basolateral surfaces, with different compositions. Proteins can be sent directly from the trans-Golgi network (TGN) to either surface, or can be sent first to one surface and then transcytosed to the other. The glycosyl phosphatidylinositol anchor is a signal for apical targeting. Signals in the cytoplasmic domain containing a β-turn determine basolateral targeting and retrieval, and are related to other sorting signals. Transcytosed proteins, such as the polymeric immunoglobulin receptor (plgR), are endocytosed from the basolateral surface and then accumulate in a tubular compartment concentrated underneath the apical surface. This compartment, tentatively termed the apical recycling compartment, may be a central sorting station, as it apparently receives material from both surfaces and sorts them for delivery to the correct surface. Delivery to the apical surface from both the TGN and the apical recycling compartment appears to be regulated by protein kinases A and C, and endocytosis from the apical surface is also regulated by kinases. Transcytosis of the plgR is additionally regulated by phosphorylation of the plgR and by ligand binding to the plgR. Regulation of traffic in polarized epithelial cells plays a central role in cellular homeostasis, response to external signals and differentiation.     

TGN38 is maintained in the trans-Golgi network by a tyrosine-containing motif in the cytoplasmic domain.

Sorting of proteins destined for different plasma membrane domains, lysosomes and secretory pathways takes place in the trans-Golgi network (TGN). TGN38 is an integral membrane protein found in this intracellular compartment. We show that TGN38 contains an autonomous targeting signal within its cytoplasmic domain which determines its intracellular location. Deletion analysis and site-directed mutagenesis of this domain demonstrate that a tyrosine motif homologous to the internalization signal of surface receptors is necessary and sufficient for correct localization. These findings suggest that TGN38 is maintained in the TGN by retrieval from the plasma membrane and employs a different mechanism for retention from that of the transferase enzymes of the trans-Golgi.     

Importance of the carboxyl-terminal FTSL motif of membrane cofactor protein for basolateral sorting and endocytosis. Positive and negative modulation by signals inside and outside the cytoplasmic tail.

Membrane cofactor protein (MCP), a widely distributed complement regulatory protein, is expressed on the basolateral surface of polarized epithelial cells, and it is not endocytosed. The carboxyl-terminal tetrapeptide (FTSL) is required for polarized surface expression. The ability of this tetrapeptide to serve as an autonomous sorting signal has been analyzed by adding this sequence motif to the C terminus of an apical membrane protein, the influenza A virus hemagglutinin (HA). The recombinant protein HA-FTSL retained the apical localization of the parental HA protein. Substitution of the complete cytoplasmic tail of MCP for the cytoplasmic tail of HA resulted in the targeting of the chimeric protein (HA/MCP) to the basolateral surface suggesting that the carboxyl-terminal FTSL motif is a weak sorting signal that requires additional targeting information from the membrane-proximal part of the cytoplasmic tail of MCP for redirecting an apical protein to the basolateral membrane domain. In contrast to the native HA, the HA-FTSL protein was subject to endocytosis. The basolateral HA/MCP was also found to be internalized and thus differed from the basolateral MCP. This result suggests that the carboxyl-terminal FTSL motif serves as an internalization signal and that in native MCP sorting information outside the cytoplasmic tail counteracts this endocytosis signal. Substitution of a tyrosine for the phenylalanine dramatically increased the internalization with most of the HA-YTSL protein being present intracellularly. Our results are consistent with the view that the interplay of multiple sorting signals and the modification of a well known targeting signal (YTSL) by amino acid exchange (FTSL) determine the constitutive expression of MCP on the basolateral surface of polarized epithelial cells.     

The targeting of Lamp1 to lysosomes is dependent on the spacing of its cytoplasmic tail tyrosine sorting motif relative to the membrane

Lamp1 is a type I transmembrane glycoprotein that is localized primarily in lysosomes and late endosomes. Newly synthesized molecules are mostly transported from the trans-Golgi network directly to endosomes and then to lysosomes. A minor pathway involves transport via the plasma membrane. The 11-amino acid cytoplasmic tail of lamp1 contains a tyrosine-based motif that has been previously shown to mediate sorting in the trans-Golgi network and rapid internalization at the plasma membrane. We studied whether this motif also mediates sorting in endosomes. We found that mutant forms of lamp1 in which all the amino acids of the cytoplasmic tail were modified except for the RKR membrane anchor and the YXXI sorting motif still localized to dense lysosomes, indicating that the YXXI motif is sufficient to confer proper intracellular targeting. However, when the spacing of the YXXI motif relative to the membrane was changed by deleting one amino acid or adding five amino acids, lysosomal targeting was almost completely abolished. Kinetic studies showed that these mutants were trapped in a recycling pathway, involving trafficking between the plasma membrane and early endocytic compartments. These findings indicate that the YXXI signal of lamp1 is recognized at several sorting sites, including the trans-Golgi network, the plasma membrane, and the early/sorting endosomes. Small changes in the spacing of this motif relative to the membrane dramatically impair sorting in the early/sorting endosomes but have only a modest effect on internalization at the plasma membrane. The spacing of sorting signals relative to the membrane may prove to be an important determinant in the functioning of these signals.     

Trafficking of immunoglobulin receptors in epithelial cells:signals and cellular factors

A typical feature of epithelial cells is the polarized distribution of their respective plasma membrane proteins. Apical and basolateral proteins can be sorted both in the trans-Golgi network and endosomes, or in both locations. Inclusion into basolateral carriers in the TGN requires the presence of distinct cytoplasmic determinants, which also appear to be recognized in endosomes. Inactivation of the basolateral sorting information leads to the efficient apical delivery, probably due to the unmasking of a recessive apical signal. Factors associated with the cytosolic face of organelles probably not only recognize these signals to mediate the inclusion of the proteins into the correct transport vesicles, but also target the carriers to the corresponding plasma membrane domain. Our interest has focused on analyzing at the molecular level how epithelial MDCK cells generate and maintain a polarized phenotype, taking advantage of immunoglobulin receptors to study the biosynthetic and endocytic pathways and the corresponding sorting events.     

TGN38 recycles basolaterally in polarized Madin-Darby canine kidney cells.

Sorting of newly synthesized plasma membrane proteins to the apical or basolateral surface domains of polarized cells is currently thought to take place within the trans-Golgi network (TGN). To explore the relationship between protein localization to the TGN and sorting to the plasma membrane in polarized epithelial cells, we have expressed constructs encoding the TGN marker, TGN38, in Madin-Darby canine kidney (MDCK) cells. We report that TGN38 is predominantly localized to the TGN of these cells and recycles via the basolateral membrane. Analyses of the distribution of Tac-TGN38 chimeric proteins in MDCK cells suggest that the cytoplasmic domain of TGN38 has information leading to both TGN localization and cycling through the basolateral surface. Mutations of the cytoplasmic domain that disrupt TGN localization also lead to nonpolarized delivery of the chimeric proteins to both surface domains. These results demonstrate an apparent equivalence of basolateral and TGN localization determinants and support an evolutionary relationship between TGN and plasma membrane sorting processes.     

Direct interaction of the trans-Golgi network membrane protein, TGN38, with the F-actin binding protein, neurabin.

TGN38 is a type I integral membrane protein that constitutively cycles between the trans-Golgi network (TGN) and plasma membrane. The cytosolic domain of TGN38 interacts with AP2 clathrin adaptor complexes via the tyrosine-containing motif (-SDYQRL-) to direct internalization from the plasma membrane. This motif has previously been shown to direct both internalization and subsequent TGN targeting of TGN38. We have used the cytosolic domain of TGN38 in a two-hybrid screen, and we have identified the brain-specific F-actin binding protein neurabin-I as a TGN38-binding protein. We demonstrate a direct interaction between TGN38 and the ubiquitous homologue of neurabin-I, neurabin-II (also called spinophilin). We have used a combination of yeast two-hybrid and in vitro protein interaction assays to show that this interaction is dependent on the serine (but not tyrosine) residue of the known TGN38 trafficking motif. We show that TGN38 interacts with the coiled coil region of neurabin in vitro and binds preferentially with the dimeric form of neurabin. TGN38 and neurabin also interact in vivo as demonstrated by coimmunoprecipitation from stably transfected PC12 cells. These data suggest that neurabin provides a direct physical link between TGN38-containing membranes and the actin cytoskeleton.     

Basolateral sorting of furin in MDCK cells requires a phenylalanine-isoleucine motif together with an acidic amino acid cluster

Furin is a subtilisin-related endoprotease which processes a wide range of bioactive proteins. Furin is concentrated in the trans-Golgi network (TGN), where proteolytic activation of many precursor proteins takes place. A significant fraction of furin, however, cycles among the TGN, the plasma membrane, and endosomes, indicating that the accumulation in the TGN reflects a dynamic localization process. The cytosolic domain of furin is necessary and sufficient for TGN localization, and two signals are responsible for retrieval of furin to the TGN. A tyrosine-based (YKGL) motif mediates internalization of furin from the cell surface into endosomes. An acidic cluster that is part of two casein kinase II phosphorylation sites (SDSEEDE) is then responsible for retrieval of furin from endosomes to the TGN. In addition, the acidic EEDE sequence also mediates endocytic activity. Here, we analyzed the sorting of furin in polarized epithelial cells. We show that furin is delivered to the basolateral surface of MDCK cells, from where a significant fraction of the protein can return to the TGN. A phenylalanine-isoleucine motif together with the acidic EEDE cluster is required for basolateral sorting and constitutes a novel signal regulating intracellular traffic of furin.