Breakdown of the Reciprocal Stabilization of QBRICK Frem1

Breakdown of the reciprocal stabilization of QBRICK͞Frem1, Fras1, and Frem2 at the basement membrane provokes Fraser syndrome-like defects Daiji Kiyozumi*†, Nagisa Sugimoto*, and Kiyotoshi Sekiguchi*†‡ *Sekiguchi Biomatrix Signaling Project, Exploratory Research for Advanced Technology, Japan Science and Technology Agency, Aichi Medical University, Nagakute, Aichi 480-1195, Japan; and †Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan Edited by Kathryn V. Anderson, Sloan–Kettering Institute, New York, NY, and approved June 19, 2006 (received for review February 6, 2006) An emerging family of extracellular matrix proteins characterized Qbrick͞Frem1 have been recognized as the genes mutated in bl, by 12 consecutive CSPG repeats and the presence of Calx-␤ motif(s) my, eb, and heb mice, respectively (4–9). Grip1 encodes an includes Fras1, QBRICK͞Frem1, and Frem2. Mutations in the genes intracellular adaptor protein containing multiple PDZ domains, encoding these proteins have been associated with mouse models whereas Fras1, Qbrick͞Frem1, and Frem2 encode members of a of Fraser syndrome, which is characterized by subepidermal blis- novel family of ECM proteins characterized by 12 consecutive tering, cryptophthalmos, syndactyly, and renal dysmorphogenesis. CSPG repeats and a varying number of Calx-␤ domains (refs. 8 Here, we report that all of these proteins are localized to the and 10; Fig. 1A). basement membrane, and that their basement membrane local- The observation of overlapping developmental defects in the ization is simultaneously impaired in Fraser syndrome model mice. four blebbing mutant mice (i.e., bl, my, eb, and heb) implies that In Frem2 mutant mice, not only Frem2 but Fras1 and QBRICK͞Frem1 the deficits underlying Fraser syndrome-like phenotypes all were depleted from the basement membrane zone. This coordi- affect a common process or pathway, in which the four Fraser nated reduction in basement membrane deposition was also ob- syndrome-associated proteins function cooperatively. Indeed, a served in another Fraser syndrome model mouse, in which GRIP1, functional linkage was shown between GRIP1 and Fras1; GRIP1 a Fras1- and Frem2-interacting adaptor protein, is primarily af- binds to the PDZ-binding motif of Fras1, which is necessary for fected. Targeted disruption of Qbrick͞Frem1 also resulted in di- the extracellular localization of Fras1 to the basal surface of minished expression of Fras1 and Frem2 at the epidermal basement epidermal cells (7). Despite conspicuous features in their domain membrane, confirming the reciprocal stabilization of QBRICK͞ structures and extracellular localization, the functions of three Frem1, Fras1, and Frem2 in this location. When expressed and Fraser syndrome-associated ECM proteins, Fras1, Frem2, and secreted by transfected cells, these proteins formed a ternary QBRICK͞Frem1, are poorly understood. It remains unclear complex, raising the possibility that their reciprocal stabilization at whether three ECM proteins function cooperatively. the basement membrane is due to complex formation. Given the In this study, we examined the cooperativity between the three close association of Fraser syndrome phenotypes with defective Fraser syndrome-associated proteins at the basement mem- epidermal–dermal interactions, the coordinated assembly of three brane. Using immunohistochemical analyses of three Fraser Fraser syndrome-associated proteins at the basement membrane syndrome model mice, including a strain of Qbrick͞Frem1Ϫ͞Ϫ appears to be instrumental in epidermal–dermal interactions dur- mice, we demonstrated that these ECM proteins fail to assemble ing morphogenetic processes. into the basement membrane when their simultaneous expression is compromised; the reciprocal expression of Fras1, Frem2, epithelial–mesenchymal interaction ͉ gene targeting ͉ morphogenesis and QBRICK͞Frem1 is required for their stable localization at the basement membrane. Failure of cooperativity between these BIOLOGY he extracellular matrix (ECM) is an insoluble supramolecu- proteins at the basement membrane explains the overlapping DEVELOPMENTAL Tlar complex surrounding metazoan cells that is often fibrous phenotypes observed in Fraser syndrome model mice. or sheet-like. The ECM functions in the control of cellular behaviors, including migration, proliferation, and differentia- Results tion, and mediates intercellular communication, as in epithelial– Diminished Expression of Frem2 in my Mutant Mice. We first exam- mesenchymal interactions; both of these functions are critical ined whether Frem2 localizes to the basement membrane in a during development. Because individual ECM components of- manner similar to Fras1 and QBRICK͞Frem1. Using an anti- ten function in combination with other ECM components and body specific to the Frem2 ectodomain, we found that Frem2 soluble factors, genetic disorders of the ECM are often linked to immunoreactivity colocalized at the epidermal basement mem- severe developmental abnormalities. brane with that of laminin-␥1, a ubiquitous marker of the Fraser syndrome is a recessive multiorgan disorder character- basement membrane (Fig. 1B). An antibody specific for the ized by cryptophthalmos, syndactyly, renal agenesis, and a cytoplasmic tail of Frem2 failed to detect the protein in situ (Fig. variety of morphogenetic defects (1). Approximately 45% of 1C), likely because of loss of the epitope after ectodomain human cases are stillborn or die within the first year, primarily shedding. In support of this possibility, Frem2 and Fras1 were because of pulmonary and͞or renal complications (2). The both secreted into the medium when expressed in mammalian phenotypic similarities between these patients and five mouse cells; the secreted Frem2 lacked a cytoplasmic tail (Fig. 1 D and ‘‘blebbing’’ mutants, blebbed (bl), myelencephalic blebs (my), eye blebs (eb), head blebs (heb), and fetal haematoma, suggested these mutant mice represent animal models of Fraser syndrome (3). Conflict of interest statement: No conflicts declared. The developmental defects observed in Fraser syndrome and the This paper was submitted directly (Track II) to the PNAS office. associated mouse models suggest that these defects arise from Abbreviations: ECM, extracellular matrix; En, embryonic day n. disruption of the epithelial–mesenchymal interactions required Data deposition: The sequence reported in this paper has been deposited in the GenBank for normal morphogenetic processes. Recently, two novel genes, database (accession no. AB236662). FRAS1 and FREM2, have been identified as the causative genes ‡To whom correspondence should be addressed. E-mail: [email protected]. in human Fraser syndrome, and Fras1, Frem2, Grip1, and © 2006 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0601011103 PNAS ͉ August 8, 2006 ͉ vol. 103 ͉ no. 32 ͉ 11981–11986 Downloaded by guest on September 24, 2021 embryos and newborns, Frem2 immunoreactivity was reduced in the epidermal basement membrane zone when compared with my͞ϩ animals (Fig. 2 B–D). Expression of other ECM proteins, including laminin-␥1, collagen-VI, NG2, collagen-IV, and per- lecan, at the epidermal basement membrane zone was unaffected (Fig. 2 H–K). Consistent with the reduction of Frem2 at the basement membrane, the expression levels of the Frem2 transcript were greatly reduced in my͞my mice from those seen in wild-type mice, although the expression of genes adjacent to Frem2 was unaffected (Fig. 2A; see also Fig. 7, which is published as supporting information on the PNAS web site). These results are consistent with recent reports that Frem2 is dysfunctional in mice bearing the myKST and myF11 alleles, both of which cause Fraser syndrome-like phenotypes similar to those observed in my mice (5, 9). Because we failed to detect any deletion or missense͞ nonsense mutation in the exons encoding the Frem2 protein (see Table 1, which is published as supporting information on the PNAS web site), the reduced expression of Frem2 may result from either mutation(s) affecting the activity of cis- transcriptional elements or the stability of Frem2 transcripts. Impaired Basement Membrane Localization of Fras1 and Frem2 in my and eb Mice. Of the proteins associated with Fraser syndrome, both Fig. 1. Localization of Frem2 at the basement membrane zone. (A) Sche- Fras1 and Frem2 contain a transmembrane domain and a PDZ matic view of the protein family that contains 12 CSPG repeats and variable domain-binding motif at their C termini (Fig. 1A); in addition, both ␤ Calx- motifs. Underlined are the regions used as immunogenic epitopes. (B are expressed by embryonic epidermal cells (refs. 5, 6, and 9; see and C) Immunolocalization of Frem2 in the skin of E17.5 mice. In the dorsal epidermal basement membrane, which is counterstained for laminin-␥1 (red), also Fig. 8, which is published as supporting information on the Frem2 (green) was detected by antibodies recognizing the ectodomain (B) but PNAS web site). Coordinated expression of Fras1 and Frem2 in not the cytoplasmic tail (C). (Scale bar, 20 ␮m.) (D and E) Secretion of Frem2 and epidermal cells suggests they are deposited concomitantly, poten- Fras1 after expression in 293F cells. 293F cells were transfected with Frem2 tially functioning in the basement membrane cooperatively. In expression vector or empty vector, followed by immunoblotting of the con- support of this possibility, Fras1 expression at the epidermal ditioned medium (M) and cell lysates (C) with

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