ATP-binding cassette transporter A1 contains an NH2-terminal signal anchor sequence that translocates the protein's first hydrophilic domain to the exoplasmic space

Mutations in the ATP-binding cassette transporter A1 (ABCA1) transporter are associated with Tangier disease and a defect in cellular cholesterol efflux. The amino terminus of the ABCA1 transporter has two putative in-frame translation initiation sites, 60 amino acids apart. A cluster of hydrophobic amino acids form a potentially cleavable signal sequence in this 60-residue extension. We investigated the functional role of this extension and found that it was required for stable protein expression of transporter constructs containing any downstream transmembrane domains. The extension directed transporter translocation across the ER membrane with an orientation that resulted in glycosylation of amino acids immediately distal to the signal sequence. Neither the native signal sequence nor a green fluorescent protein tag, fused at the amino terminus, was cleaved from ABCA1. The green fluorescent protein fusion protein had efflux activity comparable with wild type ABCA1 and demonstrated a predominantly plasma membrane distribution in transfected cells. These data establish a requirement for the upstream 60 amino acids of ABCA1. This region contains an uncleaved signal anchor sequence that positions the amino terminus in a type II orientation leading to the extracellular presentation of an approximately 600-amino acid loop in which loss-of-function mutations cluster in Tangier disease patients.     

Cloning of human and rat ABCA5/Abca5 and detection of a human splice variant

We presently report the cloning of cDNA sequences encoding the novel rat ATP-binding cassette (ABC) transporter Abca5 and the orthologous human transporter, recently designated as ABCA5. Furthermore, the existence of a novel non-translated exon of the ABCA5 gene, previously assigned to an ABCA gene cluster in the chromosomal region 17q24.2-3, is demonstrated. Abca5 and ABCA5 cDNAs are predicted to give rise to proteins of 1642 amino acids which exhibit the typical domain arrangement of ABC full transporters and share 90% identity within the amino acid sequences. A cDNA representing an ABCA5 mRNA splice variant was cloned which would result in a truncated protein equivalent to an ABC half transporter. Northern blot analyses revealed expression of ABCA5 or Abca5 mRNA in several tissues, but particularly high Abca5 mRNA expression was observed in rat testis. Up-regulation of Abca5 mRNA expression during culture of primary rat hepatocytes suggests that hepatocyte cultures should provide a basis for investigation of Abca5 gene regulation and elucidation of Abca5 function.     

ATP-binding cassette transporter A1 contains a novel C-terminal VFVNFA motif that is required for its cholesterol efflux and ApoA-I binding activities

The stimulation of cellular cholesterol and phospholipid efflux by apolipoprotein A-I is mediated by the activity of the ATP-binding cassette transporter A1 (ABCA1). Individuals with Tangier disease harbor loss-of-function mutations in this transporter that have proven useful in illuminating its activity. Here, we analyze a mutation that deletes the last 46 residues of the 2261 amino acid transporter (Delta46) and eliminates its lipid efflux. As the final four amino acids of the C terminus represent a putative PDZ-binding motif, we initially characterized deletion mutants lacking only these residues. Although a moderate decline in lipid efflux was detected, this decline was not as profound as that seen in the Delta46 mutant. Subsequent systematic analysis of the ABCA1 C terminus revealed a novel, highly conserved motif (VFVNFA) that was required for lipid efflux. Alteration of this motif, which is present in some but not all members of the ABCA family, did not prevent trafficking of the transporter to the plasma membrane but did eliminate its binding of apoA-I. Chimeric transporters, generated by substituting the C termini of either ABCA4 or ABCA7 for the endogenous terminus, demonstrated that ABCA1 could stimulate cholesterol efflux without its PDZ-binding motif but not without the VFVNFA motif. When a peptide containing the VFVNFA sequence was introduced into ABCA1-expressing cells, ABCA1-mediated lipid efflux was also markedly inhibited. These results indicate that the C-terminal VFVNFA motif of ABCA1 is essential for its lipid efflux activity. The data also suggest that this motif participates in novel protein-protein interactions that may be shared among members of the ABCA family.     

Molecular and functional interaction of the ATP-binding cassette transporter A1 with Fas-associated death domain protein

ATP-binding cassette transporter A1 (ABCA1) is a major regulator of cellular cholesterol and phospholipid homeostasis. Its function has not been fully characterized and may depend on the association with additional proteins. To identify ABCA1-interacting proteins a human liver yeast two-hybrid library was screened with the 144 C-terminal amino acids of ABCA1. Fas-associated death domain protein (FADD) was identified to bind to ABCA1, and this interaction was confirmed by pull-down assays and co-immunoprecipitations. Recombinant expression of a dominant negative form of FADD or the C terminus of ABCA1 in the human hepatoma cell line HepG2 markedly reduced the transfer of phospholipids to apoA-I. This indicates that the binding of additional proteins, one of them being full-length FADD, is required for ABCA1 function. The association of FADD with ABCA1 provides an unexpected link between high density lipoprotein metabolism and an adaptor molecule mainly described in death receptor signal transduction.     

Mutational analysis of human hydroxysteroid sulfotransferase SULT2B1 isoforms reveals that exon 1B of the SULT2B1 gene produces cholesterol sulfotransferase,whereas exon 1A yields pregnenolone sulfotransferase

As a result of an alternative exon 1, the gene for human hydroxysteroid sulfotransferase (SULTB1) encodes for two peptides differing only at their amino termini. The SULT2B1b isoform preferentially sulfonates cholesterol. Conversely, the SULT2B1a isoform avidly sulfonates pregnenolone but not cholesterol. The outstanding structural feature that distinguishes the SULT2B1 isoforms from the prototypical SULT2A1 isozyme is the presence of extended amino- and carboxyl-terminal ends in the former. Investigating the functional significance of this unique characteristic reveals that removal of 53 amino acids from the relatively long carboxyl-terminal end that is common to both SULT2B1 isoforms has no effect on the catalytic activity of either isoform. On the other hand, removal of 23 amino acids from the amino-terminal end that is unique to SULT2B1b results in loss of cholesterol sulfotransferase activity, whereas removal of 8 amino acids from the amino-terminal end that is unique to SULT2B1a has no effect on pregnenolone sulfotransferase activity. Deletion analysis along with site-directed mutagenesis of SULT2B1b reveal that the amino acid segment 19-23 residues from the amino terminus and particularly isoleucines at positions 21 and 23 are crucial for cholesterol catalysis. In the gene for SULT2B1, exon 1B encodes for only the unique amino-terminal region of SULT2B1b; however, exon 1A encodes for the unique amino-terminal end of SULT2B1a plus an additional 48 amino acids. Thus, if the gene for SULT2B1 employs exon 1B, cholesterol sulfotransferase is synthesized, whereas if exon 1A is used, pregnenolone sulfotransferase is produced.     

GLUT-4 NH2 terminus contains a phenylalanine-based targeting motif that regulates intracellular sequestration

Expression of chimeras, composed of portions of two different glucose transporter isoforms (GLUT-1 and GLUT-4), in CHO cells had indicated that the cytoplasmic NH2 terminus of GLUT-4 contains important targeting information that mediates intracellular sequestration of this isoform (Piper, R. C., C. Tai, J. W. Slot, C. S. Hahn, C. M. Rice, H. Huang, D. E. James. 1992. J. Cell Biol. 117:729-743). In the present studies, the amino acid constituents of the GLUT-4 NH2-terminal targeting domain have been identified. GLUT-4 constructs containing NH2- terminal deletions or alanine substitutions within the NH2 terminus were expressed in CHO cells using a Sindbis virus expression system. Deletion of eight amino acids from the GLUT-4 NH2 terminus or substituting alanine for phenylalanine at position 5 in GLUT-4 resulted in a marked accumulation of the transporter at the plasma membrane. Mutations at other amino acids surrounding Phe5 also caused increased cell surface expression of GLUT-4 but not to the same extent as the Phe5 mutation. GLUT-4 was also localized to clathrin lattices and this colocalization was abolished when either the first 13 amino acids were deleted or when Phe5 was changed to alanine. To ascertain whether the targeting information within the GLUT-4 NH2-terminal targeting domain could function independently of the glucose transporter structure this domain was inserted into the cytoplasmic tail of the H1 subunit of the asialoglycoprotein receptor. H1 with the GLUT-4 NH2 terminus was predominantly localized to an intracellular compartment similar to GLUT- 4 and was sequestered more from the cell surface than was the wild-type H1 protein. It is concluded that the NH2 terminus of GLUT-4 contains a phenylalanine-based targeting motif that mediates intracellular sequestration at least in part by facilitating interaction of the transporter with endocytic machinery located at the cell surface.     

Identification of a novel human sterol-sensitive ATP-binding cassette transporter (ABCA7)

We report the identification of the full-length cDNA for a novel ATP-binding cassette (ABC) transporter from human macrophages. The mRNA is of 6.8 kb size and contains an open reading frame encoding a polypeptide of 2146 amino acids with a calculated molecular weight of 220 kDa. The predicted protein product is composed of two transmembrane domains and two nucleotide binding folds indicating that it pertains to the group of full-size ABC transporters. The novel transporter shows highest protein sequence homology with the recently cloned human cholesterol and phospholipid exporter ABCA1 (54%) and the human retinal transporter ABCR (49%), both members of the ABC transporter subfamily A. In accordance with the currently proposed classification, the novel transporter was designated ABCA7. ABCA7 mRNA was detected predominantly in myelo-lymphatic tissues with highest expression in peripheral leukocytes, thymus, spleen, and bone marrow. Expression of ABCA7 is induced during in vitro differentiation of human monocytes into macrophages. In macrophages, both the ABCA7 mRNA and protein expression are upregulated in the presence of modified low density lipoprotein and downregulated by HDL(3). Our results suggest a role for ABCA7 in macrophage transmembrane lipid transport.     

Isoforms,Expression, Glycosylation,and Tissue Distribution of CTL2/SLC44A2

Antibodies to the solute carrier protein, CTL2/SLC44A2, cause hearing loss in animals, are frequently found in autoimmune hearing loss patients, and are implicated in transfusion-related acute lung injury. We cloned a novel CTL2/SLC44A2 isoform (CTL2 P1) from inner ear and identified an alternate upstream promoter and exon 1a encoding a protein of 704 amino acids which differs in the first 10–12 amino acids from the known exon 1b isoform (CTL2 P2; 706 amino acids). The expression of these CTL2/SLC44A2 isoforms, their posttranslational modifications in tissues and their localization in HEK293 cells expressing rHuCTL2/SLC44A2 were assessed. P1 and P2 isoforms with differing glycosylation are variably expressed in cochlea, tongue, heart, colon, lung, kidney, liver and spleen suggesting tissue specific differences that may influence function in each tissue. Because antibodies to CTL2/SLC44A2 have serious pathologic consequences, it is important to understand its distribution and modifications. Heterologous expression in X. laevis oocytes shows that while human CTL2-P1 does not transport choline, human CTL2-P2 exhibits detectable choline transport activity.     

Comparison of the functional insulin binding epitopes of the A and B isoforms of the insulin receptor

The human insulin receptor is expressed as two isoforms that are generated by alternate splicing of its mRNA; the B isoform has 12 additional amino acids (718-729) encoded by exon 11 of the gene. The isoforms have been reported to have different ligand binding properties. To further characterize their insulin binding properties, we have performed structure-directed alanine-scanning mutagenesis of a major insulin binding site of the receptor, formed from the receptor L1 domain (amino acids 1-470) and amino acids 705-715 at the C terminus of the alpha subunit. Alanine mutants of each isoform were transiently expressed as recombinant secreted extracellular domain in 293 cells, and their insulin binding properties were evaluated by competitive binding assays. Mutation of Arg(86) and Phe(96) of each isoform resulted in receptors that were not secreted. The Kds of unmutated receptors were almost identical for both isoforms. Several new mutations compromising insulin binding were identified. In L1, mutation of Leu(37) decreased affinity 20- to 40-fold and mutations of Val(94), Glu(97), Glu(120), and Lys(121) 3 to 10-fold for each isoform. A number of mutations produced differential effects on the two isoforms. Mutation of Asn(15) in the L1 domain and Phe(714) at the C terminus of the alpha subunit inactivated the A isoform but only reduced the affinity of the B isoform 40- to 60-fold. At the C terminus of the alpha subunit, mutations of Asp(707), Val(713), and Val(715) produced 7- to 16-fold reductions in affinity of the A isoform but were without effect on the B isoform. In contrast, alanine mutations of Tyr(708) and Asn(711) inactivated the B isoform but only reduced the affinities of the A isoform 11- and 6-fold, respectively. In conclusion, alanine-scanning mutagenesis of the insulin receptor A and B isoforms has identified several new side chains contributing to insulin binding and indicates that the energetic contributions of certain side chains differ in each isoform, suggesting that different molecular mechanisms are used to obtain the same affinity.     

Molecular structure of a novel cholesterol-responsive A subclass ABC transporter,ABCA9

We recently identified a novel ABC A subclass transporter, ABCA6, in human macrophages. Here, we report the molecular cloning of an additional ABC A subfamily transporter from macrophages denoted ABCA9. The identified coding sequence is 4.9 kb in size and codes for a 1624 amino acid protein product. In accordance with the proposed nomenclature, the novel transporter was designated ABCA9. The putative full-length ABC transporter polypeptide consists of two transmembrane domains and two nucleotide binding folds and thus conforms to the group of full-size ABC transporters. We identified alternative ABCA9 mRNA variants in human macrophages that predict the existence of three truncated forms of the novel transporter. Among the human ABC A subfamily transporters, ABCA9 exhibits the highest amino acid sequence homology with ABCA8 (72%) and ABCA6 (60%), respectively. The striking amino acid sequence similarity between these transporter molecules supports the notion that they represent an evolutionary more recently emerged subgroup within the family of ABC A transporters, which we refer to as "ABCA6-like transporters." ABCA9 mRNA is ubiquitously expressed with the highest mRNA levels in heart, brain, and fetal tissues. Analysis of the genomic structure revealed that the ABCA9 gene consists of 39 exons that are located within a genomic region of approximately 85 kb size on chromosome 17q24.2. In human macrophages, ABCA9 mRNA is induced during monocyte differentiation into macrophages and suppressed by cholesterol import indicating that ABCA9, like other known ABC A subfamily transporters, is a cholesterol-responsive gene. Based on this information, ABCA9 is likely involved in monocyte differentiation and macrophage lipid homeostasis.     

Function of the neuron-specific alternatively spliced isoforms of nonmuscle myosin II-B during mouse brain development

We report that the alternatively spliced isoforms of nonmuscle myosin heavy chain II-B (NHMC II-B) play distinct roles during mouse brain development. The B1-inserted isoform of NMHC II-B, which contains an insert of 10 amino acids near the ATP-binding region (loop 1) of the myosin heavy chain, is involved in normal migration of facial neurons. In contrast, the B2-inserted isoform, which contains an insert of 21 amino acids near the actin-binding region (loop 2), is important for postnatal development of cerebellar Purkinje cells. Deletion of the B1 alternative exon, together with reduced expression of myosin II-B, results in abnormal migration and consequent protrusion of facial neurons into the fourth ventricle. This protrusion is associated with the development of hydrocephalus. Restoring the amount of myosin II-B expression to wild-type levels prevents these defects, showing the importance of total myosin activity in facial neuron migration. In contrast, deletion of the B2 alternative exon results in abnormal development of cerebellar Purkinje cells. Cells lacking the B2-inserted isoform show reduced numbers of dendritic spines and branches. Some of the B2-ablated Purkinje cells are misplaced in the cerebellar molecular layer. All of the B2-ablated mice demonstrated impaired motor coordination.