|Coordinate||57,402,841 bp (GRCm38)|
|Base Change||T ⇒ A (forward strand)|
|Gene Name||adhesion G protein-coupled receptor E1|
|Synonym(s)||Emr1, EGF-TM7, F4/80, DD7A5-7, TM7LN3, Ly71|
|Chromosomal Location||57,358,686-57,483,529 bp (+)|
|MGI Phenotype||Strain: 3582333
FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] This gene encodes a protein that has a domain resembling seven transmembrane G protein-coupled hormone receptors (7TM receptors) at its C-terminus. The N-terminus of the encoded protein has six EGF-like modules, separated from the transmembrane segments by a serine/threonine-rich domain, a feature reminiscent of mucin-like, single-span, integral membrane glycoproteins with adhesive properties. Multiple alternatively spliced transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jan 2012]
PHENOTYPE: Homozygous null mice fail to develop peripheral tolerance after inoculation with antigen because of a lack of efferent regulatory T cell development. [provided by MGI curators]
|Amino Acid Change||Leucine changed to Stop codon|
|Institutional Source||Beutler Lab|
|Gene Model||predicted gene model for protein(s): [ENSMUSP00000004850] [ENSMUSP00000083971]|
AA Change: L166*
|Predicted Effect||probably null|
AA Change: L166*
|Predicted Effect||probably null|
|Meta Mutation Damage Score||0.9755|
|Is this an essential gene?||Possibly nonessential (E-score: 0.272)|
|Candidate Explorer Status||CE: no linkage results|
Linkage Analysis Data
|Alleles Listed at MGI|
|Mode of Inheritance||Autosomal Semidominant|
|Local Stock||Live Mice, gDNA|
|Last Updated||2017-12-19 10:33 AM by Anne Murray|
|Record Created||2013-03-07 5:19 PM by Ming Zeng|
The Onion phenotype was initially identified among G3 mice homozygous for mutations induced by N-ethyl-N-nitrosourea (ENU) and tested in the FACS screen of peripheral blood. Onion mice exhibited loss of F4/80+ macrophages in the peripheral blood (Figure 1).
|Nature of Mutation|
Whole exome HiSeq sequencing identified 89 mutations in the G1 mouse (R0049). Three G3 siblings with the Onion phenotype were genotyped at all 89 mutation sites and three mutations on chromosome 17 were homozygous in all three mice. Of these, the mutation in Emr1 was presumed to be causative because the Onion phenotype mirrored the F4/80 phenotype attributed to Emr1. Capillary sequencing confirmed a T to A transversion at 57542264 bp on Chromosome 17 (v37) in the GenBank genomic region NC_000083 encoding Emr1. The mutation corresponds to cDNA residue 497 in exon 5 of 23 total exons in transcript ENSMUST00000004850. The mutation also corresponds to cDNA residue 537 in transcript ENSMUST00000086763 (residue 537 in the mRNA transcript NM_010130.4, which corresponds to this Ensembl transcript) in exon 5 of 22 total exons.
The mutated nucleotide is indicated in red. The mutation results in a leucine (L) to premature stop codon substitution at residue 166, and may cause nonsense mediated decay of the transcript.
Emr1 encodes the F4/80 glycoprotein, which is a cell surface receptor that contains seven transmembrane (TM7) domains (Figure 3). F4/80 is a member of the B2 class of TM7 receptors, which are characterized by a long N-terminal extracellular region. Following a signal peptide, the extracellular N-terminal third of F4/80 (amino acids 32-367) contains seven tandem EGF-like domains (1), approximately 50 amino acids in length and characterized by the presence of six cysteine residues positioned to form three disulfide bonds within each domain. The EGF-like domains mediate protein-protein interactions. Five of the seven EGF-like domains of F4/80 contain consensus Ca2+-binding motifs. The C-terminal third of F4/80 (amino acids ~645-931) contains seven transmembrane segments, three extracellular loops, and three intracellular loops, with the C-terminus of the protein located intracellularly. Between the EGF-like domains at the N-terminus and the TM7 structure at the C-terminus, the middle third, or stalk region, of F4/80 has no significant similarity to other known protein domains. This portion of the protein (amino acids ~399-642) contains 4 potential N-glycosylation sites and 47 potential O-glycosylation sites (i.e. approximately 20% serine/threonine content). The Onion mutation results in coding of a premature stop codon at position 166 within the extracellular N-terminal third of F4/80 (amino acids 32-367). The Onion mutation is expected to alter both of the protein-coding transcripts of Emr1 (ENSMUST00000004850 and ENSMUST00000086763); ENSMUST00000086763 is not listed as a transcript by NCBI.
Please see the record for F4/80 for more information about Emr1.
F4/80 is best known as a macrophage-specific marker. The physiological function of F4/80 has only begun to be investigated, and evidence of its function remains sparse. F4/80-/- mice are healthy and fertile, and display no gross abnormalities or phenotypes (2;3). Histological analysis showed that immune tissues appear normal, and flow cytometric analysis demonstrated normal populations of B and T cells. Furthermore, F4/80-/- macrophage development, as determined by examination of expression profiles of several macrophage-restricted cell surface proteins, is comparable to that of wild type mice. F4/80-/- macrophages phagocytose target cells normally, and produce normal levels of nitric oxide and cytokines stimulated by lipopolysaccharide (LPS) or IFN-γ (2). F4/80-/- mice (and the ENU-induced Emr1F4/80/F4/80 mutants) also display normal resistance to infection by Listeria monocytogenes (3). This finding contrasts with previous work demonstrating that treatment with anti-F4/80 mAb impairs splenic macrophage production of tumor necrosis factor (TNF)-α and interleukin (IL)-12 induced by exposure to heat-killed Listeria (4). However, whether Listeria infection modulates the expression of F4/80 is unknown, as is the molecular effect of anti-F4/80 mAb treatment on cells.
F4/80 protein and mRNA expression have not been examined in the Onion mice, the Onion mutation is proposed to alter the surface expression of F4/80, leading to the phenotype observed in the FACS screen.
|Primers||Primers cannot be located by automatic search.|
Onion genotyping is performed by amplifying the region containing the mutation using PCR, followed by sequencing of the amplified region to detect the single nucleotide change. The following primers are used for PCR amplification:
Primers for PCR amplification
Onion(F): 5’- TCTTTGGTGGTCAGCACACAGC -3’
Onion(R): 5’- TCCTGAAGTGTGACTTGATGTGCC -3’
Primers for sequencing
Onion_seq(F): 5’- TGGTCAGCACACAGCACATAG -3’
1) 94°C 2:00
2) 94°C 0:30
3) 57°C 0:30
4) 72°C 1:00
5) repeat steps (2-4) 29x
6) 72°C 7:00
7) 4°C ∞
The following sequence of 414 nucleotides (from Genbank genomic region NC_000083 for linear DNA sequence of Emr1) is amplified:
44000 t ctttggtggt cagcacacag cacatagttc actcactctc
44041 ttttactgcc ctgcagatgt tgatgagtgt ctgacaattg ggatctgccc taagtattcc
44101 aactgctcta actctgtggg aagctacagc tgtacctgtc aaccaggctt tgtcttgaat
44161 ggctccattt gtgaaggttt gcaacacctg gtccttgcaa tttttctttt tttttcttat
44221 ttatttattt atttatttat ttatttattt atttatttat ttattctatt tatatcccaa
44281 tatcagttcc tctattcccc ccagtcattc tcttacacag ctcctccctc tattccacca
44341 tcttctcctc tgaggaggca gaggcccaac ttgtgtatca ccccaccatg gcacatcaag
44401 tcacacttca gga
Primer binding sites are underlined; sequencing primers are highlighted; the mutated nucleotide is highlighted in red.
1. McKnight, A. J., Macfarlane, A. J., Dri, P., Turley, L., Willis, A. C., and Gordon, S. (1996) Molecular Cloning of F4/80, a Murine Macrophage-Restricted Cell Surface Glycoprotein with Homology to the G-Protein-Linked Transmembrane 7 Hormone Receptor Family. J Biol Chem. 271, 486-489.
2. Lin, H. H., Faunce, D. E., Stacey, M., Terajewicz, A., Nakamura, T., Zhang-Hoover, J., Kerley, M., Mucenski, M. L., Gordon, S., and Stein-Streilein, J. (2005) The Macrophage F4/80 Receptor is Required for the Induction of Antigen-Specific Efferent Regulatory T Cells in Peripheral Tolerance. J Exp Med. 201, 1615-1625.
3. Schaller, E., Macfarlane, A. J., Rupec, R. A., Gordon, S., McKnight, A. J., and Pfeffer, K. (2002) Inactivation of the F4/80 Glycoprotein in the Mouse Germ Line. Mol Cell Biol. 22, 8035-8043.
|Science Writers||Anne Murray|
|Authors||Ming Zeng, Kuan-wen Wang, and Bruce Beutler|