Alternative titles; symbols
HGNC Approved Gene Symbol: AMFR
Cytogenetic location: 16q13 Genomic coordinates (GRCh38) : 16:56,361,452-56,425,545 (from NCBI)
| Location | Phenotype |
Phenotype MIM number |
Inheritance |
Phenotype mapping key |
|---|---|---|---|---|
| 16q13 | Spastic paraplegia 89, autosomal recessive | 620379 | Autosomal recessive | 3 |
The AMFR gene encodes a RING-H2 finger E3 ubiquitin ligase anchored at the endoplasmic reticulum (ER), where it mediates polyubiquitination of diverse substrates, including the cholesterol metabolism regulatory proteins HMGCR (142910) and INSIG1 (602055) in a process called ER-associated degradation (ERAD) (summary by Deng et al., 2023).
AMFR was initially described as a 78-kD internalizing cell surface receptor for autocrine motility factor (AMF; 172400), a protein secreted by tumor cells that stimulates tumor motility (Watanabe et al., 1991).
Watanabe et al. (1991) cloned AMFR, which they called gp78, from a fibrosarcoma cDNA library. The gene encodes a 323-amino acid polypeptide that has a single transmembrane domain and several putative glycosylation sites. The protein sequence has some homology to human tumor protein p53 (191170).
Shimizu et al. (1999) cloned full-length cDNAs for mouse and human AMFR. Both deduced proteins contain 643 amino acids, and they share 94.7% amino acid identity. AMFR has a 7-transmembrane domain topology, a RING-H2 motif, a leucine zipper motif, a potential N-glycosylation site, and several potential O-glycosylation sites. Northern blot analysis detected a 3.5-kb Amfr transcript in mouse heart, brain, lung, liver, skeletal muscle, kidney, and testis, but not in spleen.
AMFR has 4 relevant transcripts: the main transcript is isoform c, a 643-amino acid protein with a molecular weight of 73 kD comprising several functional domains (summary by Deng et al., 2023).
Silletti et al. (1993) examined the localization of AMFR on normal, papilloma, and carcinoma bladder tissue cell lines. Immunofluorescence revealed that AMFR is distributed evenly across the membrane of normal cells, has a polar cap distribution in papilloma cells, and is discretely localized to the leading and trailing edges of carcinoma cells. Only the carcinoma cells had increased motility in response to AMF.
Huang et al. (1995) found that expression of AMFR appeared to be regulated by cell-cell contact in normal fibroblasts.
Hirono et al. (1996) used immunohistochemistry to examine the expression of AMFR in gastric cancer specimens. The level of expression was associated with the pathologic stage and grade of tumor penetration. Positive AMFR expression was significantly associated with poor prognosis.
Sterol-regulated ubiquitination is an obligatory step in endoplasmic reticulum-associated degradation (ERAD) of HMG-CoA reductase (HMGCR; 142910), a rate-limiting enzyme in cholesterol synthesis. Song et al. (2005) found that rodent Gp78, a membrane-bound E3 ubiquitin ligase, associated with Insig1 (602055). Insig1 bound the membrane domain of Gp78 in the absence or presence of sterols, and upon addition of sterols, the reductase was recruited to the complex. Knockdown of Gp78 by RNA interference prevented sterol-dependent ubiquitination and degradation of endogenous reductase. Vcp (601023), an ATPase that participates in postubiquitination steps of ERAD and is required for reductase degradation, indirectly associated with Insig1 by binding Gp78. The results identified GP78 as a ubiquitin ligase that initiates sterol-dependent degradation of HMG-CoA reductase, and INSIG1 as the bridge between GP78/VCP and the reductase substrate.
Tsai et al. (2007) found that GP78 had a causal role in metastasis of an aggressive human sarcoma and that its prometastatic activity required its E3 activity. Furthermore, GP78 associated with and targeted the transmembrane metastasis suppressor KAI1 (CD82; 600623) for degradation. Suppression of GP78 increased KAI1 abundance and reduced the metastatic potential of tumor cells, an effect that was largely blocked by concomitant suppression of KAI1. Tsai et al. (2007) confirmed an inverse relationship between GP78 and KAI1 in human sarcoma tissue by microarray analysis.
By immunoprecipitation analysis, Ballar et al. (2007) showed that SVIP (620965) formed a trimeric complex with VCP and derlin-1 (DERL1; 608813). VCP and derlin-1 are also common interacting partners of GP78, but formation of the 2 complexes was mutually exclusive. By interacting with VCP and derlin-1, SVIP inhibited interaction of GP78 with CD3-delta (CD3D; 186790), VCP, and derlin-1, resulting in inhibition of CD3-delta ubiquitination and subsequent loading to VCP for retrotranslocation. The results suggested that SVIP is an endogenous inhibitor of ERAD that acts by regulating assembly of the GP78/VCP/derlin-1 complex.
Ying et al. (2009) showed that GP78 interacted with both SOD1 (147450) and ataxin-3 (ATXN3; 607047). Overexpression of GP78 promoted the ubiquitination and degradation of these 2 proteins, whereas knockdown of GP78 stabilized them. Moreover, GP78 repressed aggregate formation of mutant SOD1 and protected cells against mutant SOD1-induced cell death. Furthermore, GP78 was increased in cells transfected with these 2 mutant proteins as well as in ALS mice. Ying et al. (2009) suggested that GP78 may function in the regulation of SOD1 and ataxin-3 to target them for endoplasmic reticulum-associated degradation.
Mutation of alpha-1 antitrypsin (AAT, or SERPINA1; 107400) results in AAT deficiency (A1ATD; 613490), a disorder characterized by early-onset emphysema and liver disease. Using AT01 human liver cells overexpressing GP78, Khodayari et al. (2017) showed that GP78 regulated degradation of ubiquitinated AAT carrying the most common A1ATD-causing mutation, glu342 to lys (E342K; 107400.0011), also referred to as the Z allele, or ZAAT. GP78 targeted ZAAT for proteasomal degradation, and GP78 overexpression significantly decreased the level of accumulated ZAAT and caused faster ZAAT clearance from the ER, without significant changes in the level of extracellular ZAAT. This process involved VCP, as GP78 interacted with VCP, and the interaction was enhanced in A1ATD liver tissue. SVIP expression was relatively high in A1ATD, and overexpression of SVIP negatively regulated GP78/VCP-mediated ERAD. Knockdown analysis in AT01 cells confirmed that endogenous SVIP negatively regulated GP78 function and inhibited ZAAT degradation. Electron microscopic analysis revealed that SVIP overexpression caused vacuole formation in AT01 cells, which was abrogated by silencing of SVIP. The authors proposed that GP78 overexpression or SVIP suppression may eliminate the toxic gain of function associated with ZAAT polymerization, thus providing a novel therapeutic approach to treatment of A1ATD.
Huang et al. (1995) cloned the genomic DNA containing the 5-prime flanking region of the AMFR gene and characterized the promoter elements. The authors found that the region overlapping the transcription site is a newly identified functional transcription initiator element (Inr) that is responsible for the majority of expression of the AMFR gene.
Silletti et al. (1993) used fluorescence in situ hybridization to map the AMFR gene to human chromosome 16q21.
By interspecific backcross analysis, Shimizu et al. (1999) mapped the mouse Amfr gene to a region of chromosome 8 that shares homology of synteny with human chromosome 16q21.
Stumpf (2023) mapped the AMFR gene to chromosome 16q13 based on an alignment of the AMFR sequence (GenBank AF124145) with the genomic sequence (GRCh38).
In 20 patients from 8 unrelated consanguineous families of various origins (Morocco, Egypt, Jordan, Pakistan, and Syria) with autosomal recessive spastic paraplegia-89 (SPG89; 620379), Deng et al. (2023) identified homozygous loss-of-function mutations in the AMFR gene (see, e.g., 603243.0001-603243.0005). The patients were ascertained through international collaboration and the GeneMatcher program; the mutations were found by whole-exome, whole-genome, and targeted Sanger sequencing. All were absent from gnomAD, except 1 (W85X), which was found once in the heterozygous state. Fibroblasts derived from patients of 2 families (1 and 8) showed complete absence of the main 73-kD AMFR isoform. If expressed, the truncated AMFR proteins would lack important functional domains, causing ERAD dysfunction. AMFR-null neural stem cells showed downregulation of lipogenic genes and increased cholesterol synthesis, likely due to the stabilization of HMGCR (142910). There was altered lipid metabolism with increased numbers of enlarged lipid droplets; these defects could be rescued by expression of wildtype AMFR. Patient-derived fibroblasts and carrier fibroblasts also showed enlarged lipid droplets in a dose-dependent manner. Although there was no evidence of ER stress, electron microscopic studies showed abnormally dilated ER morphology in mutant cells. These data suggested that altered lipid metabolism and loss of ERAD function are part of the disease mechanism (see ANIMAL MODEL).
Deng et al. (2023) found that CRISPR-Cas9-mediated knockdown of the amfra gene in zebrafish resulted in smaller length and dysregulation of lipid homeostasis. There was increased lipid content, downregulation of lipogenic gene expression, and increased cholesterol synthesis. Electron microscopy showed dilated ER morphology. Amfra-null zebrafish demonstrated impaired touch-evoked escape responses associated with decreased axonal branching. Hmgcr (142910) was expected to be stabilized by the absence of amfra. Treatment of amfra-null embryos with statins (simvastatin and atorvastatin) resulted in increased length and rescued the abnormal motor behavior. Atorvastatin fully corrected axon branching defects observed in the amfra-null larvae.
In 2 brothers, born of consanguineous Moroccan parents (family 1), with autosomal recessive spastic paraplegia-89 (SPG89; 620379), Deng et al. (2023) identified a homozygous 1-bp deletion (c.12delG, NM_001323512.1) in exon 1 of the AMFR gene, predicted to result in a frameshift and premature termination (Phe5SerfsTer45). The mutation, which was found by whole-genome sequencing, segregated with the disorder in the family. The mutation was not present in the gnomAD database. Western blot analysis of patient fibroblasts showed complete absence of the main 73-kD AMFR isoform and reduced expression in heterozygous parents. There was no evidence of nonsense-mediated mRNA decay (possibly due to multiple isoforms), but even if a truncated protein were expressed, it would affect key domains important for E3 ubiquitin ligase activity and impair ERAD, resulting in a loss of function. The patients had mild global developmental delay requiring special education.
In 3 sisters, born of consanguineous Egyptian parents (family 2), with autosomal recessive spastic paraplegia-89 (SPG89; 620379), Deng et al. (2023) identified a homozygous c.254G-A transition (c.254G-A, NM_001323512.1) in the AMFR gene, resulting in a trp85-to-ter (W85X) substitution. The mutation, which was found by whole-genome sequencing, segregated with the disorder in the family. It was found once in heterozygosity in the gnomAD database (1 in 76,036 genomes). Functional studies of the variant and studies of patient cells were not performed, but it was predicted to result in a loss of function. Two of the patients had learning disabilities.
In 6 patients from a large consanguineous family from Jordan (family 3) with autosomal recessive spastic paraplegia-89 (SPG89; 620379), Deng et al. (2023) identified a homozygous c.369G-A transition (c.369G-A, NM_001323512.1) in the AMFR gene, resulting in a trp123-to-ter (W123X) substitution. The mutation, which was found by exome sequencing and targeted Sanger sequencing, segregated with the disorder in the family. It was not present in the gnomAD database. Functional studies of the variant and studies of patient cells were not performed, but it was predicted to result in a loss of function. Two of the patients had learning disabilities.
In 3 sibs, born of consanguineous Pakistani parents (family 5), with autosomal recessive spastic paraplegia-89 (SPG89; 620379), Deng et al. (2023) identified a homozygous 4-bp duplication (c.871_874dupAGCC, NM_001323512.1), predicted to result in a frameshift and premature termination (Leu292GlnfsTer14). The mutation, which was found by whole-genome sequencing, segregated with the disorder in the family. It was not present in the gnomAD database. Functional studies of the variant and studies of patient cells were not performed, but it was predicted to result in a loss of function. Two of the patients were noted to have developmental delay.
In 2 sibs, born of consanguineous Egyptian parents (family 8), with autosomal recessive spastic paraplegia-89 (SPG89; 620379), Deng et al. (2023) identified an intragenic deletion within the AMFR gene, resulting in the deletion of exons 9 and 10 (chr16.56,385,545_56,389,473del, GRCh38), and causing a frameshift and premature termination (Asn362LysfsTer22). The mutation, which was found by exome sequencing, segregated with the disorder in the family. It was not present in the gnomAD database. Western blot analysis of patient fibroblasts showed complete absence of the main 73-kD AMFR isoform and reduced expression in heterozygous parents. If expressed, the truncated protein would lack key C-terminal domains important for E3 ubiquitin ligase activity and impair ERAD, resulting in a loss of function. One of the patients had a learning disability.
Ballar, P., Zhong, Y., Nagahama, M., Tagaya, M., Shen, Y., Fang, S. Identification of SVIP as an endogenous inhibitor of endoplasmic reticulum-associated degradation. J. Biol. Chem. 282: 33908-33914, 2007. [PubMed: 17872946] [Full Text: https://doi.org/10.1074/jbc.M704446200]
Deng, R., Medico-Salsench, E., Nikoncuk, A., Ramakrishnan, R., Lanko, K., Kuhn, N. A., van der Linde, H. C., Lor-Zade, S., Albuainain, F., Shi, Y., Yousefi, S., Capo, I., and 40 others. AMFR dysfunction causes autosomal recessive spastic paraplegia in human that is amenable to statin treatment in a preclinical model. Acta Neuropath. 146: 353-368, 2023. [PubMed: 37119330] [Full Text: https://doi.org/10.1007/s00401-023-02579-9]
Hirono, Y., Fushida, S., Yonemura, Y., Yamamoto, H., Watanabe, H., Raz, A. Expression of autocrine motility factor receptor correlates with disease progression in human gastric cancer. Brit. J. Cancer 74: 2003-2007, 1996. [PubMed: 8980404] [Full Text: https://doi.org/10.1038/bjc.1996.667]
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Khodayari, N., Wang, R. L., Marek, G., Krotova, K., Kirst, M., Liu, C., Rouhani, F., Brantly, M. SVIP regulates Z variant alpha-1 antitrypsin retro-translocation by inhibiting ubiquitin ligase gp78. PLoS One 12: e0172983, 2017. [PubMed: 28301499] [Full Text: https://doi.org/10.1371/journal.pone.0172983]
Shimizu, K., Tani, M., Watanabe, H., Nagamachi, Y., Niinaka, Y., Shiroishi, T., Ohwada, S., Raz, A., Yokota, J. The autocrine motility factor receptor gene encodes a novel type of seven transmembrane protein. FEBS Lett. 456: 295-300, 1999. [PubMed: 10456327] [Full Text: https://doi.org/10.1016/s0014-5793(99)00966-7]
Silletti, S., Yao, J., Sanford, J., Mohammed, A. N., Otto, T., Wolman, S. R., Raz, A. Autocrine motility factor receptor in human bladder carcinoma: gene expression, loss of cell-contact regulation and chromosomal mapping. Int. J. Oncol. 3: 801-807, 1993. [PubMed: 21573434] [Full Text: https://doi.org/10.3892/ijo.3.5.801]
Song, B.-L., Sever, N., DeBose-Boyd, R. A. Gp78, a membrane-anchored ubiquitin ligase, associates with Insig-1 and couples sterol-regulated ubiquitination to degradation of HMG CoA reductase. Molec. Cell 19: 829-840, 2005. [PubMed: 16168377] [Full Text: https://doi.org/10.1016/j.molcel.2005.08.009]
Stumpf, A. M. Personal Communication. Baltimore, Md. 05/25/2023.
Tsai, Y. C., Mendoza, A., Mariano, J. M., Zhou, M., Kostova, Z., Chen, B., Veenstra, T., Hewitt, S. M., Helman, L. J., Khanna, C., Weissman, A. M. The ubiquitin ligase gp78 promotes sarcoma metastasis by targeting KAI1 for degradation. Nature Med. 13: 1504-1509, 2007. [PubMed: 18037895] [Full Text: https://doi.org/10.1038/nm1686]
Watanabe, H., Carmi, P., Hogan, V., Raz, T., Silletti, S., Nabi, I. R., Raz, A. Purification of human tumor cell autocrine motility factor and molecular cloning of its receptor. J. Biol. Chem. 266: 13442-13448, 1991. [PubMed: 1649192]
Ying, Z., Wang, H., Fan, H., Zhu, X., Zhou, J., Fei, E., Wang, G. Gp78, an ER associated E3, promotes SOD1 and ataxin-3 degradation. Hum. Molec. Genet. 18: 4268-4281, 2009. [PubMed: 19661182] [Full Text: https://doi.org/10.1093/hmg/ddp380]