BIOLOGICAL OVERVIEW

Rapid flow of information in the nervous system involves presynaptic vesicle recycling by clathrin-mediated endocytosis, an event triggered by the alpha-Adaptin-containing AP2 complex. A Drosophila alpha-Adaptin is expressed in the garland cells, imaginal discs, and the CNS. In presynaptic terminals, alpha-Adaptin defines a network-like membrane structure to which the GTPase dynamin is recruited. alpha-Adaptin is necessary for the formation of clathrin-coated pits and participates in the dynamin-dependent release of coated vesicles from the membrane surface. These results suggest an alpha-adaptin-dependent control of the vesicle cycle that maintains the balance between the amount of vesicle- and surface-associated membranes (González-Gaitán, 1997).

alpha-Adaptin also acts downstream of Numb in the determination of alternative cell fates in asymmetric cell division. During asymmetric cell division in sensory organ precursor cells, Numb protein localizes asymmetrically and segregates into one daughter cell, where it influences cell fate by repressing signal transduction via the Notch receptor. Numb acts by polarizing the distribution of alpha-Adaptin, a protein involved in receptor-mediated endocytosis. alpha-Adaptin binds to Numb and localizes asymmetrically in a Numb-dependent fashion. Mutant forms of alpha-Adaptin that no longer bind to Numb fail to localize asymmetrically and cause numb-like defects in asymmetric cell division. These results suggest a model in which Numb influences cell fate by downregulating Notch through polarized receptor-mediated endocytosis, since Numb also binds to the intracellular domain of Notch (Berdnik, 2002).

Adaptins are subunits of adaptor protein (AP) complexes involved in the formation of intracellular transport vesicles and in the selection of cargo for incorporation into the vesicles. The term 'adaptin' was coined to designate a group of ~100 kDa proteins that copurify with clathrin upon isolation of clathrin-coated vesicles. The ~100 kDa-proteins are subunits of heterotetrameric adaptor protein (AP) complexes, and the term 'adaptin' has been extended to all subunits of these complexes. Four basic AP complexes have been described: AP-1, AP-2, AP-3, and AP-4. Each of these complexes is composed of two large adaptins (one each of g/alpha/d/e and ß1-4, respectively, 90-130 kDa), one medium adaptin (µ1-4, ~50 kDa), and one small adaptin (s1-4, ~20 kDa). [See the figure in the Hill review (2001) for information on the AP structure]. The analogous adaptins of the four AP complexes are homologous to one another (21-83% identity at the amino acid level). In general, the subunits of different AP complexes are not interchangeable, with the exception of some nonmammalian ß1/2 hybrid proteins, and possibly mammalian ß1 andß2, which can be components of both AP-1 and AP-2. Some of the adaptins occur as two or more closely-related isoforms encoded by different genes. Additional diversity arises from alternative splicing of adaptin mRNAs. Thus, cells that express several of these adaptin variants have the potential to assemble a diverse array of AP complexes. AP-1, AP-2, and AP-3 are expressed in all eukaryotic cells examined to date. AP-4, on the other hand, is ubiquitously expressed in man (Homo sapiens), mouse (Mus musculus), chicken (Gallus gallus), and the plant Arabidopsis thaliana, but not in the fruit fly Drosophila melanogaster, the nematode Caenorhabditis elegans, and the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe (Hill, 2001 and references therein).

AP complexes are components of protein coats that associate with the cytoplasmic face of organelles of the secretory and endocytic pathways. The complexes participate in the formation of coated vesicular carriers, as well as in the selection of cargo molecules for incorporation into the carriers. AP-2 mediates rapid endocytosis from the plasma membrane, while AP-1, AP-3, and AP-4 mediate sorting events at the trans-Golgi network (TGN) and/or endosomes. AP-1 and AP-2 function in conjunction with clathrin, whereas AP-4 is most likely part of a nonclathrin coat. Mammalian (but not yeast) AP-3 has been shown to interact with clathrin, but the functional significance of this interaction is still unclear. The AP complexes have the overall shape of a 'head' with two protruding 'ears' connected to the head by flexible 'hinge' domains (Hill, 2001 and references therein).

Synaptic transmission requires the repeated release of neurotransmitters at high frequency at the nerve terminal. The current understanding of this rapid communication process between the nerve cells and their targets is primarily based on the biochemistry of the synaptic vesicles, protein–protein interaction studies, structural analyses, electrophysiology, and imaging. These studies suggest that the repeated release of transmitters involves a synaptic vesicle cycle that includes the formation of neurotransmitter-filled vesicles, the release of the neurotransmitter by exocytosis, recycling of both the membrane and the release machinery by endocytosis, and, finally, the regeneration of vesicles that undergo a new round of exocytosis/endocytosis. Each step of the vesicle cycle involves a number of identified components, and their assembly is likely to be mediated by distinct protein–protein interactions (González-Gaitán, 1997 and references therein).

While recent studies have focused on the proteins involved in exocytosis, it is not clear whether (or to what extent) the vesicle membrane recycles via clathrin-coated vesicles, or whether the membrane is directly retrieved by a fast endocytotic process. Biochemical studies lead to the proposal that the AP2 complex, a heterotetramer containing alpha-adaptin, plays an essential role in orchestrating different steps of endocytosis at the synapse. AP2 is able to bind to the cytoplasmic tail of a number of membrane receptors including Synaptotagmin, a transmembrane protein that controls the Ca²⁺-dependent membrane fusion during exocytosis. Synaptotagmin interaction with AP2 is consistent with its proposed function during endocytosis, which is based on a synaptotagmin endocytotic mutant phenotype. This would argue that synaptotagmin plays a dual role in the vesicle cycle by acting in the final step of exocytosis and the initial step of endocytosis, thereby coupling the two processes at the plasma membrane. Once recruited to the inner surface of the plasma membrane, AP2 is likely to initiate the formation of clathrin-coated pits by triggering the assembly of clathrin triskelion subunits into a polygonal lattice that causes a bending of the membrane into the coated pit structure. Clathrin-coated pits detach from the plasmalemma by a GTP-dependent fission reaction that is mediated by the GTPase dynamin, and the resulting coated membrane vesicles become internalized. Dynamin was shown to bind the AP2 complex in vitro, and to be functionally required for the detachment of the clathrin-coated vesicles from the membrane. After internalization, the clathrin-coated vesicles shed their coats, a process that involves a number of proteins, including auxilin, Hsp-70, and the cystein string protein (CSP), which may function in a chaperone-like manner to unfold the clathrin lattice at the outer surface (González-Gaitán, 1997 and references therein).

While there is little doubt that synaptic vesicle membranes are endocytosed by clathrin-coated vesicles, it remains to be established whether this pathway is essential for synaptic transmission. The most direct evidence is derived from an analysis of the only endocytotic mutant isolated to date, the Drosophila mutant shibire (shi), which affects the gene coding for Dynamin. In a temperature-sensitive shi mutant, animals develop normally at the permissive temperature. At the restrictive temperature, however, release of transmitter ceases, leading to rapid paralysis. Examination of the shi mutant phenotype by electron microscopy indicated that endocytosis is normally initiated and clathrin-coated pits are formed but remain trapped in the collared pit stage. Thus, vesicle recycling and, consequently, transmitter release are blocked when the function of dynamin is impaired (González-Gaitán, 1997 and references therein).

Mutations in a Drosophila alpha-Adaptin gene (D-alphaAda) have been used to study recycling of synaptic vesicles. At the larval neuromuscular junction, synaptic vesicle recycling is impaired in weak alpha-Ada mutants and blocked in alpha-adaptin-deficient embryos. alpha-adaptin is confined to areas of the presynaptic plasma membrane that are distinct from the sites containing the active zones of exocytosis. Furthermore, alpha-adaptin is required for the recruitment of dynamin to the endocytotic sites (González-Gaitán, 1997).

Cloning of alphaAdaptin was initiated through a P element enhancer trap insertion found in a screen for genes expressed in the embryonic nervous system. The corresponding P element maps to cytological position 21C1-2. A genomic DNA fragment adjacent to the P element insertion site was identified and a genomic walk was constructed encompassing the region that turned out to be the alphaAdaptin transcription unit. Northern blot analysis with polyA⁺ RNA of embryos and adults revealed that alphaAdaptin codes for two transcripts of about 4.8 and 6.3 kb. Both transcripts appear with similar intensities in the polyA⁺ RNA of embryos, while in adults, the smaller transcript appears to be enriched over the longer one (González-Gaitán, 1997).

Rapid synaptic transmission in the nervous system takes advantage of the general property of cells to recycle the membrane by endocytosis. Consistently, vertebrate alpha-Adaptins have been found to be expressed ubiquitously, with the exception of an alpha-Adaptin-A isoform that is specifically enriched in neural cells. Likewise, Drosophila dynamin, which is required during synaptic transmission, is ubiquitously expressed and required. Evidence is provided for an essential role of a Drosophila alpha-Adaptin in recycling of synaptic vesicles. The highly restricted expression patterns of the Drosophila alpha-Adaptin and the specific defects in aAdaptin mutants exclude a general role for alpha-Adaptin in endocytosis in all cells. Also, the mutant defect indicates that the lack of alphaAdaptin activity in the nervous system cannot be compensated for by a redundant action of other members of the alpha-Adaptin family, which must exist in the Drosophila genome to function in endocytosis outside of the nervous system (González-Gaitán, 1997).

In vitro studies have shown that alpha-Adaptin triggers the formation of a clathrin lattice that turns into empty coat structures. This finding, and the position of AP2 between the clathrin lattice and the vesicle membrane, is consistent with the proposal that the formation of coated vesicles is initiated by an alpha-Adaptin-dependent recruitment of clathrin to the membrane. The lack of vesicles and the corresponding increase of the presynaptic plasma membrane in the aAdaptin mutant establish that alpha-Adaptin is indeed required for the formation of clathrin-coated pits. The resulting lack of membrane recycling causes an increase of the membrane surface, indicating that the pool of vesicle membranes is fused with the presynaptic plasma membrane of the alpha-Ada mutant (González-Gaitán, 1997).

At the presynaptic terminal, vesicles dock and fuse to release neurotransmitters by exocytosis. In Drosophila, active zones of transmitter release at the plasma membrane are a discrete electron-dense structure, the so-called dense bodies. At the neuromuscular junctions, such active zones are found in patterns of interspersed islands. The restricted network-like structure of alpha-Adaptin in both wild-type and shi mutant presynaptic terminals, where vesicles formation is blocked at the collared pit stage, leaves such islands void of alpha-Adaptin. This observation suggests that alpha-Adaptin defines regions within the presynaptic membrane that are complementary to the distribution of active zones. If this inference is correct, it would imply that exocytosis and endocytosis events occur in different locations at the presynaptic membrane (González-Gaitán, 1997).

The compositions of the plasma membrane and the synaptic vesicle membranes are different. This supports the argument that the membranes of exocytotic vesicles never fuse completely with the plasma membrane and instead open up at the fusion site to release the transmitter, closing immediately thereafter. Such a mechanism implies that active zones of exocytosis and the site of vesicle recycling would coincide. However, most recent ultrastructural analysis of shi mutant synaptic terminals provides evidence for two distinct pathways for vesicle re-formation. One pathway emanates from the active zone of exocytosis and has a fast time course. It involves small clusters of vesicles that are observed at the active zones. The formation of these vesicles does not include intermediate structures, such as coated pits, coated vesicles, or cisternae, and might be accomplished by a direct pinch-off at the plasma membrane. The second pathway emanates from sites away from the active zones and results in the re-formation of the rest of the vesicle population throughout the terminal. It has a slower time course and involves coated collared pits (González-Gaitán, 1997).

The distinct staining pattern of alpha-Adaptin, in a network-like array, suggests that alpha-Adaptin acts preferentially, if not exclusively, in the second pathway involving coated vesicles outside the active zones. Furthermore, the lack of vesicles and a corresponding expansion of the plasma membrane in alpha-Adaptin-deficient embryos are consistent with a recycling mechanism that builds upon a complete fusion between the vesicle and plasma membrane compartments during exocytosis, and the re-formation of vesicle membranes at separate centers of endocytosis (González-Gaitán, 1997).

Dynamin, originally identified as a microtubule-binding protein, has been shown to have GTPase activity in vitro, containing three GTP-binding consensus motifs. Introducing mutations that interfere with GTP binding has been found to have no effect on microtubules in transfected cells, but endocytosis is blocked. In shi mutants, the clathrin-coated pits are normally formed at the plasma membrane but fail to pinch off the membrane. In contrast, the coated pits fail to form in the membrane of Drosophila alpha-Adaptin mutants. These observations formally argue that alpha-Adaptin and dynamin act in two distinct steps during vesicle recycling, and they are not consistent with the proposed direct interaction between AP2 and dynamin as revealed by in vitro studies (Wang, 1995). However, an increased temperature sensitivity of the shi mutation caused by the lack of one functional copy of the alpha-Adaptin gene strongly suggests that the two molecules act in linked processes, and, therefore, that alpha-Adaptin is also required for the dynamin-dependent internalization of the vesicles. This finding, together with the subcellular localization of alpha-Adaptin in wild type and the colocalization of alpha-Adaptin and dynamin in shi mutant synapses, is therefore consistent with a function of the alpha-Adaptin-containing AP2 complex in recruiting the GTPase dynamin to the assembled clathrin-coated pits (Wang, 1995). The genetic interaction shown in this study also indicates that alpha-Adaptin directly or indirectly interacts with dynamin. This strongly suggests that alpha-Adaptin functions upstream of dynamin, since it acts at an earlier step of endocytosis than dynamin (as indicated by their respective mutant phenotypes), and since the subcellular localization of alpha-Adaptin is not affected in shi mutants. These findings indicate a molecular link between the formation of clathrin-coated pits and the detachment of coated vesicles, coordinating two consecutive steps of the process of endocytosis (González-Gaitán, 1997).

The alpha-Adaptin mutant phenotype indicates that the lack of vesicle formation affects not only physiological aspects of synapse function but also causes a corresponding increase of the plasma membrane complement. This suggests that membrane fractions present in the vesicle and plasma membrane compartments, and their rapid exchange by exocytosis/endocytosis events, are regulated to maintain the surface area of the synapse and the pool of available synaptic vesicles. This control obviously does not involve exocytosis, since it proceeds to cause an expansion of the membrane surface in the alpha-Adaptin mutant synapses. Neither is vesicle internalization the controlling step, because in the dynamin mutant shi, the membrane can be processed into clathrin-coated pits that remain attached to the plasma membrane. Furthermore, collared coated pits are rarely observed in wild type, suggesting that they represent a very transient intermediate, and, therefore, that vesicle internalization by dynamin is not a limiting step (González-Gaitán, 1997).

One can envision a scenario where the recruitment of the AP2 complex to the plasma membrane is a rate-limiting step, which in turn could be controlled by membrane-associated AP2 receptors that are released from exocytotic vesicles. Such a molecular link between exocytosis and endocytosis events would guarantee exocytosis-dependent membrane retrieval, as suggested by the temporal link of exocytosis and endocytosis and by the in vitro interaction between AP2 and synaptotagmin (Zhang, 1994). The results of this study show, however, that synaptotagmin does not colocalize with alpha-Adaptin in shi mutants. This suggests that the role of synaptotagmin and AP2 association (Zhang, 1994) is more likely to serve the recycling of synaptotagmin from the membrane, returning it to the cytoplasmic pool of synaptic vesicles to take part in the subsequent exocytosis event. Alternatively, synaptotagmin could be one of several functionally redundant receptors to anchor the AP2 complex to the membrane to initiate a new vesicle cycle (González-Gaitán, 1997).

Numb regulates cell fate by polarizing the distribution of alpha-Adaptin

Cells can use different mechanisms to generate diversity among their progeny. They can divide into two initially identical cells, which then become different by interacting with each other or with their environment. Alternatively, cell divisions can become intrinsically asymmetric when cell fate determinants are segregated into one of the two daughter cells during mitosis and establish a particular cell fate in this cell, but not in its sister cell. In the Drosophila peripheral nervous system, a combination of these two mechanisms is used to create the four different cell types found in external sensory (ES) organs. ES organs are composed of two outer cells, which differentiate into a hair and a socket, and two inner cells, which form a neuron and a sheath. During development, these four cells arise from a single sensory organ precursor (SOP) cell in a stereotyped lineage. The SOP cell first divides into a posterior pIIa and an anterior pIIb cell. Next, the pIIb cell divides, to generate the pIIIb cell and a glia cell, which migrates away and is not part of the ES organ. Finally, the pIIa cell gives rise to the hair and the socket while the pIIIb cell generates the neuron and sheath (Berdnik, 2002).

The generation of diverse cell fates in the peripheral nervous system, in the SOP lineage, requires the numb gene. In numb mutants, SOP cells divide symmetrically into two pIIa cells. Each of these, in turn, generates two socket cells, so that morphologically abnormal ES organs with four sockets are formed. Conversely, when the numb gene is overexpressed in SOP cells, the opposite cell fate transformation is observed, and, in the most extreme case, all cells are transformed into neurons. numb encodes a PTB (phosphotyrosine binding) domain protein, which is present in all cells and uniformly distributed around the cell cortex in interphase. In mitotic neural precursor cells, however, the protein concentrates in the area of the cell cortex that overlies one of the two centrosomes. Thus, Numb acts as a segregating determinant in the SOP lineage (Berdnik, 2002).

Numb influences cell fate by repressing signal transduction through the transmembrane receptor Notch. Notch is part of a conserved signaling pathway that is used many times during development. Binding of one of the two ligands, Delta or Serrate, to the extracellular domain of Notch leads to proteolytic cleavages, first in the extracellular and then in the intracellular domain. Upon cleavage, the intracellular part of Notch translocates to the nucleus, where it converts the transcription factor Suppressor of Hairless [Su(H)] from a repressor into a transcriptional activator. Analysis of Notch function during asymmetric cell division in the bristle lineage is complicated by the fact that it is also required for lateral inhibition during specification of SOP cells. In the absence of Notch, too many SOP cells are specified, leading to the generation of supernumerary bristles. However, inactivation of Notch function after SOP cell determination using temperature-sensitive alleles leads to cell fate transformations that are opposite of those observed in numb: the SOP cell divides into two pIIb cells, and, in the most extreme cases, four neurons are generated. Thus, Numb and Notch act antagonistically in specifying correct cell fate in the bristle lineage. Genetically, numb acts upstream of Notch in this process. A model has begun to emerge in which Notch is activated in both daughter cells by its ligands Delta and Serrate, which act redundantly in the SOP lineage. While Notch signaling induces the pIIa cell fate in one daughter cell, Numb prevents signal transduction in the other daughter cell and allows this cell to assume the pIIb cell fate. In vitro binding and two-hybrid experiments have suggested that Numb directly binds to the intracellular domain of Notch, but how Numb binding prevents Notch signaling has been unclear (Berdnik, 2002 and references therein).

A mammalian homolog of Numb was shown to localize to endocytic vesicles and to bind to the endocytic protein alpha-Adaptin (Santolini, 2000). alpha-Adaptin is an essential component of the AP-2 complex, a heterotetramer that functions as an adapter between the intracellular domain of transmembrane receptors destined for endocytosis and the endocytic machinery (Clague, 1998; Robinson, 1994). Recruitment of AP-2 to the plasma membrane promotes the polymerization of clathrin into a cage-like structure, the formation of coated pits, and, ultimately, the internalization of the bound transmembrane receptors. Structural analysis has revealed that AP-2 contains a brick-like core domain, which is connected by flexible linkers to two appendage domains (Heuser, 1988). These so-called ear domains are formed by the C-terminal-most 25–30 kDa regions of alpha- and ß2-Adaptin (Berdnik, 2002 and references therein).

Like its vertebrate counterpart, Drosophila alpha-Adaptin binds to Numb and the ear domain of alpha-Adaptin is critical for this interaction. Like Numb, alpha-Adaptin localizes asymmetrically in dividing SOP cells and preferentially segregates into the pIIb cell. alpha-Adaptin mutations that affect binding to Numb and abolish asymmetric localization cause cell fate transformations similar to those observed in numb. Epistasis experiments place alpha-Adaptin downstream of numb and upstream of Notch, suggesting that alpha-Adaptin is involved in the suppression of Notch signaling by Numb. These results suggest that Numb regulates cell fate by polarizing the distribution of the endocytic protein alpha-Adaptin which in turn is involved in the endocytosis and consequent inactivation of Notch (Berdnik, 2002).

alpha-Adaptin serves a dual function. During receptor-mediated endocytosis, ligand binding induces recruitment of the protein to the intracellular domain of transmembrane receptors (Notch for example). This first regulates signal transduction by targeting the receptor for endocytosis. Genetic analysis of this function during signal transduction is hampered by a second role in constitutive, ligand-independent endocytosis of certain transmembrane receptors, for example, the transferrin receptor. Constitutive endocytosis is essential for cell viability, and, therefore, alpha-Adaptin null mutant cells do not survive (Gonzalez-Gaitan, 1997). The ada^ear alleles do not affect cell viability. Several observations indicate that they are not simply hypomorphs but that they specifically affect particular functions of alpha-Adaptin during signal transduction. First, ada^ear mutants are viable over known alpha-Adaptin hypomorphs. These hypomorphs affect larval motility due to impaired synaptic transmission at the neuromuscular junction (Gonzalez-Gaitan, 1997). No such defects are observed in ada^ear mutants, suggesting that alpha-Adaptin function during synaptic transmission is not, or is only weakly, impaired. Second, ada^ear mutants have defects in asymmetric cell division, but no such defects have been described for any of the known alleles (Berdnik, 2002).

All ada^ear mutants affect the C-terminal so-called ear (or appendage) domain of the protein. This domain was shown by electron microscopy to stick out from a brick-shaped central core in the AP-2 complex (Heuser, 1988). The crystal structure of this domain (Owen, 1999) reveals an N-terminal subdomain (lysine 713–Lysine 826 in the Drosophila protein) and a C-terminal subdomain (Phenylalanine 827–Phenylalanine 940) connected by a short linker. While the C-terminal subdomain makes all the known protein interactions, the N terminus forms a scaffold that displays the C-terminal domain in the correct orientation. All ada^ear mutants affect the C-terminal subdomain: ada^ear4 and ada^ear5 introduce stops at the beginning of this subdomain. ada^ear26 deletes two amino acids in the linker between the two subdomains and might disrupt the correct spatial arrangement of the C-terminal subdomain. Thus, ada^ear mutations are predicted to generate forms of alpha-Adaptin that lack functional ear domains (Berdnik, 2002).

The experiments suggest that the ear domain is not required for alpha-Adaptin function in general endocytosis. While recent experiments have suggested a role of Adaptin ear-domains in clathrin assembly (Clairmont, 1997), the current results are more consistent with previous experiments in which removal of this domain by proteolytic cleavage did not affect the ability of AP-2 to assemble clathrin-coated pits and form endocytic vesicles in vitro (Keen, 1989; Peeler, 1993). The alpha-Adaptin ear domain was shown to bind other important endocytic proteins, like Eps-15 and Epsin (Owen, 1999), and these important interactions should be disrupted in ada^ear mutants. However, both proteins also bind to the ß2-Adaptin ear domain (Owen, 2000), and redundancy between the binding sites could explain why the mutant protein is still partially functional (Berdnik, 2002).

alpha-Adaptin is essential for Numb to repress Notch signaling in the pIIb cell. alpha-Adaptin is part of the AP-2 complex that has a well-established function in clathrin-mediated endocytosis of transmembrane receptors (Clague, 1998; Gonzalez-Gaitan; Robinson, 1994; Takei, 2001). At the moment, it cannot be excluded that the role of alpha-Adaptin in asymmetric cell division reflects a novel function of the protein that is not related to endocytosis. However, all the known functions of alpha-Adaptin are connected to endocytic processes, and, therefore, these results indicate that Numb influences cell fate by polarizing endocytosis of a critical component of the Notch pathway. Several results indicate that this critical component is the Notch receptor itself. First, alpha-Adaptin functions genetically upstream of Notch, and, therefore, proteins acting downstream of Notch are unlikely to be targets for endocytosis. Second, cell culture experiments have shown that Numb acts in the Notch expressing, signal receiving cell, and it is therefore unlikely to act on Delta or Serrate. Third, Numb can interact with the intracellular domain of Notch in vitro and in two-hybrid assays. This in vitro interaction is supported by the observation that transfection of Numb can recruit the Notch intracellular domain to the cell cortex in tissue culture cells (Berdnik, 2002 and references therein).

A model is proposed in which Numb serves as an adapter that links the AP-2 complex to the intracellular domain of Notch on one side of a dividing SOP cell. This targets the receptor for endocytosis and reduces Notch signaling in the pIIb cell. After division, reciprocal signaling between the two daughter cells can lead to competition, and small differences could be amplified by negative-feedback loops that are known to exist between cells that communicate via Notch and Delta. A slight reduction of Notch levels in one of the two cells should be enough to bias this competition and to establish the pIIb cell fate in the cell that inherits Numb. Consistent with this, differences in Notch protein levels in the two daughter cells could not be detected by immunofluorescence (Berdnik, 2002).

This model is plausible; nevertheless, alternative explanations cannot be excluded. For example, Numb could specifically act on the activated form of Notch. Upon ligand binding, proteolytic cleavage removes the extracellular domain of Notch and generates an intermediate fragment that is anchored in the plasma membrane. A second, unregulated cleavage by the transmembrane protease presenilin at the plasma membrane releases the intracellular domain, which translocates to the nucleus to activate gene transcription. alpha-Adaptin could enhance endocytosis of the intermediate fragment and target it for degradation before the second cleavage can occur. This would reduce Notch signaling without significantly affecting the total amount of Notch protein (Berdnik, 2002).

A vertebrate homolog of Numb was previously shown to bind alpha-Adaptin (Santolini, 2000). Like in flies, the ear domain is essential for this protein interaction, but the functional significance of this interaction in vertebrates is not completely understood. Fragments of Numb that could act as dominant negatives inhibit both EGF and transferrin-receptor uptake in cell culture (Santolini, 2000), suggesting a function for Numb in both constitutive and ligand-induced endocytosis. In Drosophila, however, no phenotypes are detected upon overexpression of the analogous domain. Furthermore, none of mutant phenotypes described for numb so far indicates defects in EGF-receptor signaling or cell lethality, and, therefore, a function in general endocytosis is highly unlikely. Whether these differences reflect inhibition of other pathways by the dominant negative or a true difference across species is not clear (Berdnik, 2002).

In Drosophila the known phenotypes of numb can be explained by a function in asymmetric cell division. One of the mouse Numb homologs, in contrast, is not asymmetrically localized and is unlikely to be involved in asymmetric cell division. Regulation of alpha-Adaptin-mediated endocytosis is a more likely common task of the Numb protein family. In fact, the function in endocytosis may extend to other more distant sequence homologs: the cell death protein Ced-6 and the adaptor protein ARH are the closely related to Numb (Garcia, 2001). The molecular function of Ced-6 is not understood, while ARH is mutated in familial hypercholesterinaemia and involved in endocytic uptake of the LDL receptor upon ligand binding (Garcia, 2001). Thus, Numb, Ced-6, and ARH might be members of a protein superfamily involved in endocytosis of transmembrane receptors (Berdnik, 2002).

GENE STRUCTURE

cDNA clone length - 3252 bases

Bases in 5' UTR - 399

Exons - 7

Bases in 3' UTR - 30

PROTEIN STRUCTURE

Amino Acids - 940

Structural Domains

From a library prepared from polyA⁺ RNA of 0- to 18-hr-old embryos, a 4.8 kb cDNA was isolated that is likely to represent a full-size cDNA of the smaller transcript. Northern blots with cDNA fragments indicated that the 4.8 and 6.3 kb transcripts share the leader region and the open reading frame. The physical extent of the smaller transcript was mapped by hybridization of the cDNA clone within the walk and by sequencing the cDNA and relevant portions of the genomic DNA flanking the exon/intron boundaries. Conceptual translation of the single open reading frame revealed a 940 amino acid homolog of vertebrate alpha-adaptins. Overall, the Drosophila alpha-Adaptin shows 70% and 66% amino acid identity to vertebrate alpha-adaptin-C and alpha-adaptin-A, respectively. alpha-adaptins are composed of characteristic regions that include the N-terminal head domain, the flexible hinge domain, and the ear domain. The sequence similarity between Drosophila alpha-Adaptin and the vertebrate homologs is more pronounced in the head domain than in the hinge or ear domains, as has been observed among the different vertebrate homologs (González-Gaitán, 1997).

date revised: 2 January 2023

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