Genomic structure of the human M6P/IGF2 receptor

The human mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) gene located at 6q26 (Rao et al. 1994) encodes for a multifunctional receptor that possesses distinct binding sites for phosphomannosyl glycoproteins and IGF2 (MacDonald et al. 1988; Morgan et al. 1987), retinoic acid (Kang et al. 1997), and urokinase-type plasminogen activator receptor (uPAR; Nykjaer et al. 1998). The receptor is involved in fetal development, tumor suppression, maternal regulation of intrauterine growth, and has recently been associated with the development of human cognitive ability (Chorney et al. 1998; De Souza et al. 1997; Kornfeld 1992; Wang et al. 1994). To facilitate further genetic analyses of the human M6P/IGF2R, we have determined its complete genomic organization, defined the intron-exon boundary sequences, and designed intronic oligonucleotides for PCR amplification of the 48 exons included in the 136-kb genomic sequence. The M6P/IGF2R is a chimeric receptor possessing binding sites for four distinct classes of ligands (Kang et al. 1997; Nykjaer et al. 1998; MacDonald et al. 1988; Morgan et al. 1987). The M6P binding site enables the M6P/IGF2R to target to the lysosomes both newly synthesized lysosomal enzymes from the Golgi and phosphorylated proteolytic enzymes endocytosed from the extracellular environment (Kornfeld 1992). Furthermore, the latent complex of transforming growth factor beta (TGFb), a potent growth inhibitor, binds to the M6P/IGF2R through these M6P binding sites, thereby facilitating its activation by plasmin (Dennis and Rifkin 1991). The effectiveness of TGFb activation may be further enhanced by the direct binding of uPAR to the M6P/IGF2R (Nykjaer et al. 1998). The mammalian M6P/IGF2R also contains an independent IGF2 binding site that is absent in chickens (Zhou and Sly 1995). IGF2 binding to the M6P/IGF2R leads to lysosomal degradation rather than intracellular signaling, a process mediated by both the IGF1 and the insulin receptors (Kornfeld 1992). Therefore, loss of M6P/IGF2R function would be predicted to increase the extracellular concentration of IGF2, decrease the level of active TGFb, and increase the secretion of proteolytic enzymes. These biological effects suggest the M6P/IGF2R functions normally as a tumor suppressor. A number of reports now strongly support this postulate for a wide variety of human tumors. Briefly, the M6P/IGF2R is mutated in 60% of dysplastic liver lesions and HCCs in patients with or without hepatitis virus (HV) infection (De Souza et al. 1995a, 1995b; Yamada et al. 1997). Its inactivation also plays a prominent role in the early stage of breast cancer development (Hankins et al. 1996), and increased expression of the wild-type receptor in breast cancer cell lines leads to apoptosis (Oates et al. 1998). Furthermore, the M6P/IGF2R gene contains a poly-G region that is a common mutational target in colon, gastric, and endometrial tumors with mismatch repair deficiences and microsatellite instability (Souza et al. 1996). Thus, M6P/IGF2R inactivation is a frequent oncogenic event that occurs early in carcinogenesis. A full parental complement of autosomal genes is inherited by all offspring, but not all are biallelically expressed. The phenomenon of monoallelic expression of the same parental allele is called genomic imprinting (Surani 1998). The M6p/Igf2r gene is imprinted in mice (Barlow et al. 1991) and is expressed only from the maternal allele in all tissues except potentially the brain (Hu et al. 1998). In contrast, imprinting of the M6P/IGF2R gene is a polymorphic trait in humans, with most people having biallelic expression in all tissues (Xu et al. 1993). Although imprinting of the M6P/IGF2R may have provided an evolutionary advantage to the mother during the emergence of mammalian intrauterine growth (Haig and Graham 1991), monoallelic expression of this gene would also be predicted to increase cancer susceptibility. In support of this postulate, Xu et al. and associates (1997) have recently demonstrated M6P/IGF2R imprinting in 50% of Wilm’s tumors. Plomin and his colleagues have also identified the M6P/IGF2R as a putative ‘‘IQ gene’’ (Chorney et al. 1998). By comparing Caucasian children with an IQ of 160 or higher with those with an average IQ, they showed that the M6P/IGF2R is linked to human cognitive ability. If a polymorphism in the M6P/IGF2R coding sequence or its regulatory region is subsequently shown to functionally affect IQ, it would be the first identified gene known to contribute to intelligence. Thus, the M6P/IGF2R plays a fundamental role in biological processes ranging from embryogenesis to carcinogenesis. How the M6P/IGF2R could possess such diverse biological functions is an intriguing enigma that remains to be solved. To facilitate future studies on the human M6P/IGF2R, we have characterized its complete genomic structure. The human cDNA sequence was divided into 48 exons based upon the mouse genomic structure (Szebenyi and Rotwein 1994), and PCR primers were designed to traverse these putative introns. All the introns except 1, 2, and 4 were amplified from human genomic DNA with either long-template PCR (Boehringer Mannheim Corp., Indianapolis, Ind.) or traditional PCR (Qiagen, Inc, Santa Clarita, Calif.). PCR products were sequenced on an ABI automated sequencer (Perkin-Elmer Corp., Foster City, Calif.). The sizes of introns 1 and 2 and their 58 and 38 boundary sequences were previously defined (Riesewijk et al. 1996; Smrzka et al. 1995); this information is available on GenBank (Accession Nos. X83699, X83700, and X83701). To sequence intron 4, human BAC clones (174E20 and 650K6) were first obtained by probing filters from Research Genetics, Inc. (Huntsville, Ala.) with an RT-PCR product spanning exons 1 to 6. A 13-kb stretch of DNA containing intron 4 was then PCR amplified from this BAC clone and the exon-intron boundaries sequenced. With the exception of intron 15, all exon-intron splice sites (93/94) conformed to the AG/GT rule (Mount 1982). Interestingly, the single intron splice site exception is also present in the mouse M6p/Igf2r homolog (Szebenyi and Rotwein 1994). Intron sizes were determined by sequencing the entire intron or estimated by electrophoresis. Twenty introns were sequenced entirely (that is, introns 8, 9, 13–17, 21, 23, 24, 27, 28, 32–34, 37, 38, 42, 43, and 46), and at least 100 bp of flanking intronic sequence were determined for the remaining introns. The entire human M6P/ IGF2R gene was then reconstructed with the use of the previously Correspondence to: R.L. Jirtle Mammalian Genome 10, 74–77 (1999).