The CD47 gene is 48,771 bases in size and is composed of 11 exons encoding a 5234 base mRNA (https://www.ncbi.nlm.nih.gov/nuccore/NM_198793.3) and 5 additional alternatively spliced transcripts (Figure 2) (Schnickel et al., 2002). Transcripts CD47-201 and CD47-202 encode isoforms with different C-terminal cytoplasmic tails. Splicing of alternative 3-UTRs in the transcripts control localization of newly translated CD47 proteins (Berkovits and Mayr 2015; Ma and Mayr 2018). The long UTR recruits HuR (ELAVL1) and SET and directs CD47 to the cell surface via the endoplasmic reticulum and Golgi, whereas the short UTR directs CD47 mRNA translation to intracellular TIS granules.

In the human CD47 gene, the 5 sequence from -272 to the ATGs contains binding sites for transcription factors including TFAP2A (AP-2), MAZ, CREB1, SP1, and E2F. Its expression is regulated by a α-Pal/NRF-1 region (Chang and Huang, 2004). CD47 expression is increased in many cancers with progression of disease including ovarian carcinoma, T-cell leukemia and lymphoma, and multiple myeloma (Massuger et al., 1991; Campbell et al., 1992; Raetz et al., 2005; Rendtlew Danielsen et al., 2007; Majeti et al., 2009; Willingham et al., 2012; Matlung et al., 2017; Murata et al., 2018; Logtenberg et al., 2020). Increased CD47 expression was linked to poor prognosis in many cancers (Willingham et al., 2012; Logtenberg et al., 2020). However, analysis of TCGA RNAseq data indicated that the inverse correlation between CD47 mRNA expression and survival is not universal. Increased CD47 mRNA expression in melanomas is positively associated with increased survival (Nath et al., 2019).
Increased CD47 transcription in cancer is driven by a variety of factors including MYC (Casey et al., 2016). Hypoxia-inducible factor-1 ( HIF1A) directly induces CD47 expression in breast cancer (Zhang et al., 2015). This may account, in part, for the upregulation of CD47 as cancers are poorly perfused and hypoxic. The CD47 signaling ligand THBS1 was increased in response to hypoxia in a HIF-dependent manner (Labrousse-Arias et al., 2016). Similarly, SRSF10, via mIL1RAP-NF-kB, induced CD47 expression (Liu et al., 2018). CD47 expression in cancer cells was reduced after treatment with BRAF / MAP2K7 (MEK) inhibitors (Liu et al., 2017) and the DNER (BET) inhibitor JQ1 (Li et al., 2019). Expression of mutant isocitrate dehydrogenase 1 in gliomas led to disruption of PKM - CTNNB1 (β-Catenin) - SMARCA4 (BRG1) transcriptional regulation and decreased CD47 expression (Gowda et al., 2018).
Cancer therapy can also alter CD47 expression. Multiple chemotherapeutic drugs including carboplatin, doxorubicin, gemcitabine, and paclitaxel induced CD47 expression (Samanta et al., 2018). MIR222 downregulated CD47 expression in irradiated tumour cells (Shi et al., 2019).
In contrast to cancers, CD47 was downregulated in multiple sclerosis lesions by MIR34A, MIR155 and MIR326, which target the 3 UTR of CD47 (Junker et al., 2009). MIR34A downregulated CD47 expression via PI3K/AKT and protected cells in the spinal cord from apoptosis (Qi et al., 2019).

none identified

Alternative exon splicing produces CD47 isoforms with short or long C-terminal cytoplasmic tails (Figure 2). The CD47 long isoform precursor contains 323 amino acids, has a mass of 35214 Da, and contains an 18 residue N-terminal signal peptide. The mature long isoform protein comprises residues 19-323, and the short isoform comprises residues 19-305. The N-terminal residue of the mature protein Gln-19 is enzymatically modified to pyrrolidone carboxylic acid (pyroGlu) (Logtenberg et al., 2019), which is required for binding to SIRPA. Mature CD47 is an integral membrane protein that contains an extracellular immunoglobulin domain and a transmembrane domain with 5 membrane spanning segments related to the presenilins (Figure 3). The long isoform cytoplasmic tail contains a ubiquitinylation site at Lys-317 (Kim et al., 2011). The protein contains disulfide bonds linking Cys at positions 33 to 263, which links the IgV domain to the transmembrane domain (Figure 3), and 41 to 114 within the IgV domain. Heterogeneous N-linked glycosylation is found at asparagine residues 23, 34, 50, 73, 111, and 206 (Hatherley et al., 2008; Shiromizu et al., 2013) which results in the typical diffuse migration of CD47 at 50-60 kDa on SDS gel electrophoresis. In several cell types, CD47 is further modified to an apparent of molecular mass >250 kDa by heparan and chondroitin sulfate glycosaminoglycan modification at residue Ser-64, which is required for THBS1-dependent inhibition of T cell receptor signaling (Kaur et al., 2011).

CD47 is ubiquitously expressed on hematopoietic cells including thymocytes, T and B cells, monocytes, platelets, and erythrocytes, as well as on epithelial, endothelial (Isenberg et al., 2006), vascular smooth muscle (Isenberg et al., 2007) and neural cells, platelets, fibroblasts, sperm, and tumor cell lines (Barclay et al., 1997). CD47 homozygous knockout (Cd47^-/-) mice are viable and fertile but exhibit defects in responses to some pathogens (Lindberg et al., 1996; Navarathna et al., 2015; Nath et al., 2018). Due to lack of inhibitory SIRPA signaling, wild type mice eliminated cd47^-/- RBCs via erythrophagocytosis, which identified CD47 as a marker of self commonly known as a "dont eat me" signal (Oldenborg et al., 2000). Under inflammatory conditions or infection, the cd47^-/- mice developed anemia and splenomegaly (Bian et al., 2016). Following intestinal epithelium wounding, cd47^-/- mice had delayed healing (Reed et al., 2019). In contrast, cd47^-/- mice exhibited enhanced protection from ionization radiation (Isenberg et al., 2008a), thermal injury (Soto-Pantoja et al., 2014a), ischemia, ischemia-reperfusion (Isenberg et al., 2008b), hypoxia- (Rogers et al., 2017b) and sickle cell disease-mediated (Novelli et al., 2019) pulmonary hypertension, and Fas-mediated apoptosis (Manna et al., 2005). Null animals showed enhanced vasorelaxation and blood flow via increased nitric oxide/cGMP and VEGF signaling (Isenberg et al., 2007; Kaur et al., 2010; Bauer et al., 2010). Young cd47^-/- mice had more efficient and more numerous mitochondria in certain skeletal muscles (Frazier et al., 2011), more stem cells and self-renewal capacity (Kaur et al., 2013), and global protection of anabolic metabolites (Miller et al., 2015). Increased or decreased expression of CD47 was reported in several nonmalignant diseases and is associated with pathogenesis as detailed below.
Increased expression of CD47 in malignant tissues was first reported in ovarian cancer (Massuger et al., 1991; Campbell et al., 1992), and subsequently confirmed in various solid tumors and hematologic malignancies (Matlung et al., 2017; Murata et al., 2018; Logtenberg et al., 2020; Wiersma et al., 2015). RNAseq data from the TCGA PanCancer Atlas indicated that CD47 mRNA expression is highest in human ovarian serous cystadenocarcinoma followed by uterine corpus endometrial carcinoma, lung adenocarcinoma and head and neck squamous cell carcinoma (Figure 4). The high expression of CD47 in ovarian cancers was explored as an imaging modality and strategy for targeted radiotherapy and drug delivery (Massuger et al., 1991; Lu et al., 2001). However, subsequent appreciation of the ubiquitous expression of CD47 in nonmalignant cells limited further development of this approach.
Elevated CD47 expression is a prognostic marker in some cancers, with higher CD47 protein and/or mRNA expression associated with decreased survival, which was attributed to SIRPA-dependent suppression of tumor cell phagocytosis by macrophages (Oldenborg et al., 2000; Jaiswal et al., 2009; Willingham et al., 2012). Alternatively, several studies have shown that elevated CD47 expression supports the maintenance of cancer stem/tumor initiating cells, which in turn provides a selective pressure for maintaining elevated CD47 expression (Lee at al., 2014; Kaur et al., 2016; Kaur and Roberts, 2016). However, analysis of TCGA data indicated that elevated CD47 mRNA expression has a protective function in some cancers including cutaneous melanoma (Nath et al., 2019). Elevated CD47 expression in melanoma correlated with markers of enhanced T cell and NK cell-mediated antitumor immunity.

CD47 mRNA containing the long 3 UTR is targeted to the ER where SET binds to the cytoplasmic domain and, with activation of RAC1, translocates CD47 to the plasma membrane (Berkovits and Mayr 2015). CD47 translated from mRNA containing the short UTR is targeted to a membraneless cytoplasmic intracellular compartment containing TIS granules (Ma and Mayr 2018). CD47 function may depend on its localization as expression of CD47 encoded by the short, but not the long, UTR isoform restored radiosensitivity in a CD47-deficient T cell line (Berkovits and Mayr 2015). CD47 is also present on extracellular vesicles isolated from body fluids and regulates the biological cargo and intercellular signaling function of these vesicles (Kaur et al., 2014; Kibria et al., 2016; Tong et al., 2016).

CD47 is also known as integrin associated protein based on its initial isolation by co-purification with beta-3 integrin ( ITGB3) (Lindberg et al., 1993). Ligation of CD47 regulates the activation of associated integrins and their function in mediating cell adhesion and migration (Cooper et al., 1995; Gao et al., 1996; Wang and Frazier 1998; Yoshida et al., 2000; Barazi et al., 2002; Brittain et al., 2004). Although it has only a small cytoplasmic tail, CD47 interacts with the cytoplasmic partners ubiquilin-1 and ubiquilin-2 ( UBQLN1 and UBQLN2, formerly known as PLIC1 and PLIC2) (Wu et al., 1999) and BNIP3 (Lamy et al., 2003). Ubiquilins mediate CD47 interaction with heterotrimeric G proteins containing Giα ( GNAI1) (NDiaye et al., 2003; Fujimoto et al., 2003; Frazier et al., 1999). The extracellular domain of CD47 interacts with its counter-receptor signal regulatory protein-α (SIRPA, also known as SIRPα, SHPS-1, BIT, P84), which is expressed on dendritic cells, and macrophages, in synapse-rich regions of the brain, and on numerous other cell types including endothelial cells, vascular smooth muscle cells (Han et al., 2000; Jiang et al., 1999), and renal tubular epithelial cells (Yao et al., 2014). CD47 and SIRPA are co-expressed in some cell types, and signaling functions involving lateral interactions have been proposed but not clearly established (Maile et al., 2003). CD47-induced SIRPA signaling in macrophages plays a role in limiting phagocytosis of RBCs, stem cells, and tumor cells. CD47 laterally associates with VEGFR2 ( KDR) in endothelial cells (Kaur et al., 2010) and Jurkat T cells (Kaur et al., 2014). CD47 also associates with the Rh blood group antigen complex, and deficiency of RHCE, band 4.2 ( EPB42), or RHAG proteins leads to decreased CD47 expression (Van Kim et al., 2006; Flatt et al., 2012; Cambot et al., 2013). THBS1 signaling via CD47 on T cells inhibits their activation and effector functions (Li et al., 2001; Li et al., 2002; Lamy et al., 2007; Kaur et al., 2011; Miller et al, 2013). Conversely, CD47-dependent integrin activation can co-stimulate T cell receptor signaling, and its deficiency leads to T cell anergy (Reinhold et al., 1999).
CD47 binds to the C-terminal domain of THBS1 (Brown and Frazier 2001; Isenberg et al., 2009) and mediates calcium (Schwartz et al., 1993), cAMP (Wang et al., 1999; Manna and Frazier 2003; Manna and Frazier 2002; Yao et al., 2011), and nitric oxide/cGMP signaling (Isenberg et al., 2006; Rogers et al., 2017a), hydrogen sulfide biosynthesis (Kaur et al., 2015; Soto-Pantoja et al., 2015), cell survival from ionization radiation and chemotherapeutic drugs (Kaur et al., 2019; Soto-Pantoja et al., 2015), autophagy (Soto-Pantoja et al., 2012), stem cell self-renewal (Kaur et al., 2013) and extracellular vesicle signaling (Kaur et al., 2018).
CD47 differentially regulates normal and malignant cell responses to genotoxic damage caused by ionizing radiation and chemotherapeutic drugs (Maxhimer et al., 2009; Feliz-Mosquea et al., 2018; Kaur et al., 2019). CD47 null or THBS1 null mice were highly resistant to high-dose radiation with minimal soft-tissue or bone marrow injury (Isenberg et al., 2008a). This response was mediated by an increase in protective autophagy (Soto-Pantoja et al., 2012; Feliz-Mosquea et al., 2018), anabolic metabolism, antioxidant, and DNA repair pathways (Miller et al., 2015). However, in cancers, disruption of THBS1-CD47 signaling sensitized tumors in immune-competent mice to ionizing radiation and chemotherapeutic drugs (Maxhimer et al., 2009; Feliz-Mosquea et al., 2018). This effect was mediated by activation of T and NK cell-dependent tumor killing (Soto-Pantoja et al., 2014b; Nath et al., 2019). Treatment with an oligonucleotide morpholino that blocks CD47 mRNA translation, resulting in decreased total CD47 protein, protected wild type mice from lethal whole-body radiation (Soto-Pantoja et al., 2013).
Similarly, CD47 plays divergent roles in regulation of stem cell self-renewal. Lack of CD47 in healthy tissues and cells upregulates the essential self-renewal transcription factors POU5F1 (Oct3/4), SOX2, KLF4, and MYC and increases the abundance of stem cells (Kaur et al., 2013). Conversely, in breast cancer, glioma, and hepatocellular carcinomas stem/tumor initiating cells, inhibiting CD47 signaling decreased cancer stem cell self-renewal and asymmetric cell division (Lee et al., 2014; Lo et al., 2016; Kaur et al., 2016; Kaur and Roberts 2016; Li et al., 2017a).

CD47 is a member of the immunoglobin super family that is conserved across amniotes including mammals, reptiles, and birds (https://www.ncbi.nlm.nih.gov/gene/961/ortholog/?scope=32524). The IgV domain of CD47 is distantly related to the Drosophila melanogaster wrapper (AF134113, Stork et al. 2009), a GPI-linked membrane protein involved in axon ensheathment by glia (Noordermeer et al., 1998). The transmembrane domain shares homology with the presenilins (Roberts et al., 2012). A viral ortholog of CD47 was first reported in myxoma virus (AF170726) (Cameron et al., 1999) and contributed to virulence by limiting macrophage activation during infection in rabbits (Cameron et al., 2005). CD47-related genes are present in many Poxviridae including Vaccinia virus CD47 (AAB96477), variola virus CD47 (P33853) and Yaba monkey tumor virus (AB025319). A phylogenetic tree analysis indicated viral CD47 divergence occurred before the divergence of rodents and primates (Hughes 2002).

Human CD47 polymorphisms
Polymorphisms in CD47 were linked to cancer risk and outcome for individuals with colorectal cancer (Thean et al, 2018; Lacorz et al., 2013). Fourteen instances of altered CD47 copy number with pathogenic significance were reported to date in ClinVar (https://www.ncbi.nlm.nih.gov/clinvar). In each case, multiple adjacent genes were duplicated or deleted. Thus, specific pathogenic roles for CD47 in these situations remain to be identified. The only CD47 SNP reported in the NCBI occurs in an intron (rs12695175 A/C). This SNP was related to variation in expression of CD47 and associated with immune-mediated skin cell injury in individuals with Pemphigus foliaceus (Bumiller-Bini et al., 2019). Consistent with the lack of nonsense mutations in the NCBI data, exon sequencing of over 60,000 human genomes identified only 1 putative loss-of-function mutation in CD47 versus 11.2 loss-of-function mutations predicted for a gene of its size (Lek et al., 2016) (Figure 4). Based on this, CD47 has an 89% probability of being loss intolerant. Mice lacking CD47 are viable, indicating the gene is not essential in a controlled laboratory setting. However, the impaired immune responses of cd47^-/- mice to some pathogens and dysregulation of vascular physiology and hemostasis are potential reasons for the gene to be loss-intolerant in humans (Lindberg et al., 1996; Navarathna et al., 2015; Nath et al., 2018; Soto-Pantoja et al. 2015).
Non-Human CD47 Polymorphisms
The Tibetan plateau is characterized by reduced atmospheric oxygen and increased radiation exposure. High throughput sequencing of blood samples from Tibetan plateau chickens, which reside at approximately 3,650 meters above sea level, found significant enrichment in a missense SNP of CD47 in comparison to blood samples from lowland chickens (Zhang et al., 2016). This is potentially relevant given that CD47 limits cardiovascular response to hypoxia, ischemia, ischemia reperfusion, metabolism and radiation-mediated genotoxic injury. Also, absence of CD47 leads to less oxygen utilization and improved mitochondrial function in mice (Frazier et al., 2011). It is possible the identified CD47 SNP encodes for a mutant protein with less activity and signaling to potentiate adaptation to the low oxygen/high radiation conditions of life at elevated altitude.

The incidence of somatic mutations of CD47 in human cancers is low, with a frequency of about 1.7% (The Cancer Genome Atlas, cbioprortal.org). Amplification is the most common variant (Figure 5). CD47 is sporadically amplified in lung squamous, ovarian, and cervical cancers, but rarely in other cancer types. CD47 amplification is associated with increased CD47 mRNA expression (Figure 4, lower panel). The majority of the 50 point mutations identified to date are missense or nonsense. These mutations are randomly distributed and have not revealed any cancer-specific mutation hotspots (Figure 6). Rare mutations that lead to shallow and deep deletion of CD47 are found in uterine endometrial carcinoma, sarcoma, prostate adenocarcinoma, lung squamous cell carcinoma, low grade glioma and colorectal adenocarcinoma (TCGA, PanCancer Atlas).

CD47 was upregulated in human astrocytoma cell lines. Blocking CD47 in these cells led to decreased expression of UHRF1 (Ubiquitin-like containing PHD and RING finger-1) and increased expression of the tumour suppressor gene CDKN2A (p16 or INK4A) (Boukhari et al., 2015). In multiple myeloma, CD47 expression was upregulated by treating cells with a DNA methyltransferase inhibitor and a histone deacetylase inhibitor (De Beck et al., 2018). HDAC inhibitor treatment decreased CD47⁺ leukemic cells and reversed their chemo-resistant phenotype (Yan et al., 2019). CD47 expression in lymphoma cells decreased following treatment with JQ1 (Li et al., 2019). miR-133a inhibited CD47 mRNA and protein expression in laryngeal carcinoma cells (Li et al., 2016). Conversely, in a prostate cancer cell line, CD47 epigenetically regulated the expression of Schlafen-11 ( SLFN11), which modifies of cancer cell sensitivity to DNA damaging agents (Kaur et al., 2019).