The half-life of c-myc mRNA is regulated when cells change their growth rates or differentiate. Two regions within c-myc mRNA determine its short half-life. One is in the 3′-untranslated region, the other is in the coding region. A cytoplasmic protein, the coding region determinant-binding protein (CRD-BP), binds in vitro to the c-myc coding region instability determinant. We have proposed that the CRD-BP, when bound to the mRNA, shields the mRNA from endonucleolytic attack and thereby prolongs the mRNA half-life. Here we report the cloning and further characterization of the mouse CRD-BP, a 577 amino acid protein containing four hnRNP K-homology domains, two RNP domains, an RGG RNA-binding domain and nuclear import and export signals. The CRD-BP is closely related to the chicken β-actin zipcode-binding protein and is similar to three other proteins, one of which is overexpressed in some human cancers. Recombinant mouse CRD-BP binds specifically to c-myc CRD RNA in vitro and reacts with antibody against human CRD-BP. Most of the CRD-BP in the cell is cytoplasmic and co-sediments with ribosomal subunits.

Several matrix metalloproteinases, including the 92-kDa and 72-kDa gelatinases, macrophage metalloelastase (MME), and matrilysin degrade insoluble elastin. Because elastolytically active MME and matrilysin consist only of a catalytic domain (CD), we speculated that the homologous CDs of the 92-kDa and 72-kDa gelatinases would confer their elastolytic activities. In contrast to the MME CD, the 92 and 72 CDs expressed in Escherichia coli (lacking the internal fibronectin type II-like repeats) had no elastase activity, although both were gelatinolytic and cleaved a thiopeptolide substrate at rates comparable to the full-length gelatinases. Elastin is an extracellular matrix protein composed of highly cross-linked, hydrophobic tropoelastin monomers which provides resilience to elastic fibers. The hydrophobicity and extensive cross-linking of tropoelastin monomers result in an insoluble elastic fiber which is highly resistant to proteolysis (1). Thus, under normal physiologic conditions, elastin undergoes minimal turnover (2). However, certain pathologic situations, including pulmonary emphysema (3) and abdominal aortic aneurysm (4), are characterized by proteolytic destruction of elastic fibers. The involvement of serine proteases in such pathologies has long been suspected. More recently, participation of cysteine proteinases (5) and matrix metalloproteinases (MMPs, 1 6 -8) in these diseases has been proposed. The MMPs comprise a gene family that collectively is capable of degrading all components of extracellular matrix in physiologic and pathologic states (9, 10). As presently recognized, this family consists of fibroblast (11), neutrophil (12), and breast carcinoma-derived (13) collagenases, three stromelysins, 92-kDa and 72-kDa gelatinases, macrophage metalloelastase (MME, MMP-12), matrilysin, and a recently described 66-kDa membrane-type metalloproteinase (14). These enzymes are organized into homologous structural domains, with some differences in domain composition and number. All members share a zymogen domain, containing a conserved PRCGXPD motif involved in enzyme latency, and a zinc-binding CD. Most members also contain a hemopexin-like domain at their C terminus, the exception being matrilysin, which lacks this domain completely. Unique to the 72-kDa and 92-kDa gelatinases is an additional domain composed of three fibronectin type II-like repeats inserted in tandem within the zinc-binding CD. The 92-kDa gelatinase also contains an ␣2(V) collagen-like domain not found in any of the other family members.The issue of substrate specificity has received considerable attention recently in MMP biology. The determinants which confer substrate specificity to these enzymes appear to be localized within discrete structural domains. For example, the ability of the collagenases to degrade triple-helical collagen requires the presence of the C-terminal hemopexin-like domain (15)(16)(17). In contrast, the stromelysins degrade a variety of substrates in a manner which is independent of the C-terminal hemopexin-like dom...

We have analyzed mammary tumors arising in transgenic mice expressing a novel, multifunctional RNA-binding protein. The protein, which we call the c-myc mRNA coding region instability determinant binding protein (CRD-BP), binds to c-myc, insulin-like growth factor II, and ␤-actin mRNAs, and to H19 RNA. Depending on the RNA substrate, the CRD-BP affects RNA localization, translation, or stability. CRD-BP levels are high during fetal development but low or undetectable in normal adult tissues. The CRD-BP is linked to tumorigenesis, because its expression is reactivated in some adult human breast, colon, and lung tumors. These data suggest the CRD-BP is a proto-oncogene. To test this idea, the CRD-BP was expressed from the whey acidic protein (WAP) promoter in mammary epithelial cells of adult transgenic mice. The incidence of mammary tumors was 95% and 60% in two lines of WAP-CRD-BP mice with high and low relative CRD-BP expression, respectively. Some of the tumors metastasized. Nontransgenic mice did not develop mammary tumors. H19 RNA and insulin-like growth factor II mRNA were up-regulated significantly in non-neoplastic WAP-CRD-BP mammary tissue. WAP-CRD-BP mice are a novel model for mammary neoplasia and might provide insights into human breast cancer biology.

C57BL/6 (B6) and DBA/2 (D2) mice exhibit disparate behavior when tested for voluntary morphine intake in a two-bottle choice drinking paradigm with B6 mice consuming 10 times more drug than D2 mice. Previous genetic mapping studies identified a locus, Mop2, on the proximal part of chromosome 10 that explained over half of the genetic variance in this mouse model of opioid selfadministration. We constructed a set of reciprocal congenic strains between B6 and D2 mice in which the proximal portion of chromosome 10 has been introgressed from one strain onto the background of the other. We tested mice from this pair of reciprocal strains together with progenitor B6 and D2 mice in a two-bottle choice drinking paradigm with morphine and quinine. The results showed that introgression of chromosome 10 alleles from the B6 strain onto a D2 genetic background increased voluntary morphine intake four-fold compared to progenitor D2 mice. Preference for morphine was also increased significantly in D2.B6-Mop2 mice compared to progenitor D2 mice. Conversely, introgression of chromosome 10 alleles from the D2 strain onto a B6 genetic background decreased morphine intake by half compared to progenitor B6 mice in B6.D2 -Mop2 mice; however, high morphine preference was maintained in this congenic strain most likely due to strong quinine aversion. When quinine was eliminated from the control bottle, morphine preference in B6.D2-Mop2 mice was decreased significantly relative to B6 and D2.B6-Mop2 mice. Overall, these data confirm the existence of a gene(s) on chromosome 10 proximal to D10Mit124 that has a strong influence on the difference in morphine drinking behavior between B6 and D2 mice.