Molecular Cloning of POEM

Abstract

Cell adhesion molecules are involved in a number of biological functions, such as cell survival, cell differentiation, tissue repair, and development. A novel molecule, POEM (preosteoblast epidermal growth factor-like repeat protein with meprin, A5 protein, and receptor protein-tyrosine phosphatase domain), was isolated by reverse transcription-polymerase chain reaction using a set of degenerate primers designed after other known epidermal growth factor (EGF)-like motifs. From its structure, POEM was suggested to be a novel adhesion molecule with five EGF-like domains, an Arg-GlyAsp (RGD) cell binding motif, and a meprin, A5 protein, and receptor protein-tyrosine phosphatase (MAM) domain. By in situ hybridization using embryonic day 16.5 (E16.5) mouse embryos, strong expression of POEM mRNA was observed in developing kidney renal tubules, parathyroid and thyroid glands, developing bone, tooth germ, and endocrine organs of the brain. The inner ear, skeletal muscle, smooth muscle (except for the vascular system), and skin were also positive for POEM expression. Bacterial recombinant POEM protein containing the RGD sequence and MAM domain showed strong cell adhesion, spreading, and survival-promoting activities. By mutating the RGD sequence to RGE, the cell spreading and survival activities were significantly decreased, but the MAM domain was shown to contribute only to cell adhesion and not to cell spreading and survival-promoting activities. The distribution of POEM in several tissues was close to that of ␣ 8 ␤ 1 integrin. Therefore, we conducted cell adhesion assays using KA8 cells, a K562 leukemia clone stably expressing ␣ 8 integrin. Parental K562 cells, which expressed ␣ 5 ␤ 1 integrin, bound to fibronectin but not to POEM. On the other hand, KA8 cells showed strong binding and spreading on both fibronectin and POEM. These results suggest that POEM is a novel ligand for ␣ 8 ␤ 1 integrin and that POEM may be involved in the development and function of various tissues, such as kidney, bone, muscles, and endocrine organs.

Molecular Basis of the Recognition of Nephronectin by Integrin α8β1

“…Purified nephronectin gave a broad band at ϳ120 kDa under reducing conditions, which was significantly larger than the predicted protein size (61 kDa). The apparent discrepancy was probably caused by modification with a large number of O-linked sugar chains, which has been predicted to occur at the mucin-like region within the central linker segment (10,11). The recombinant nephronectin gave a less prominent band at ϳ120 kDa upon immunoblotting with an anti-FLAG mAb under non-reducing conditions (Fig.…”

“…Disruption of the ␣8 gene in mice was found to be associated with severe defects in kidney morphogenesis (5) and stereocilia development (6). To date, ␣8␤1 integrin has been shown to bind to fibronectin, vitronectin, osteopontin, latency-associated peptide of transforming growth factor-␤1, tenascin-W, and nephronectin (also named POEM) (7)(8)(9)(10)(11)(12)(13), among which nephronectin is believed to be an ␣8␤1 integrin ligand involved in kidney development (10).…”

“…Nephronectin is one of the basement membrane proteins whose expression and localization patterns are restricted in a tissue-specific and developmentally regulated manner (10,11). Nephronectin consists of five epidermal growth factor-like repeats, a linker segment containing the RGD cell-adhesive motif (designated RGD-linker) and a meprin-A5 protein-receptor protein-tyrosine phosphatase (MAM) domain (see Fig.…”

Expression of nephronectin is enhanced by 1α,25‐dihydroxyvitamin D3

“…Nephronectin (Npnt), an extracellular matrix protein, is known to be involved in the development and functions of various tissues 1, 2. This protein, which acts as an adhesion molecule, consists of five epidermal growth factor (EGF)‐like domains, an Arg‐Gly‐Asp (RGD) cell binding motif, and a meprin A5 protein and receptor protein‐tyrosine phosphatase μ (MAM) domain 1, 2.…”

“…This protein, which acts as an adhesion molecule, consists of five epidermal growth factor (EGF)‐like domains, an Arg‐Gly‐Asp (RGD) cell binding motif, and a meprin A5 protein and receptor protein‐tyrosine phosphatase μ (MAM) domain 1, 2. Npnt interacts with integrins, especially α8β1 integrins, and plays a crucial role during the early steps of kidney morphogenesis through its own RGD motif 2.…”