We investigated the influence of the activation state of integrin ␣5␤1 on its dependence on the PHSRN synergy site for binding to RGD in fibronectin. K562 and MV3 cells lacked ␣v␤3 expression and adhered to fibronectin through ␣5␤1. Mel57 cells adhered through ␣v␤3 and ␣5␤1. A recombinant fibronectin polypeptide, containing five type III repeats from the central cell binding domain 3Fn6 -10, and a mutated polypeptide lacking the synergy site were equally effective in promoting Mel57 adhesion. For K562 and MV3, the mutated polypeptide was not or poorly active compared to the control polypeptide. Expression of ␣v␤3 in MV3 induced strong adhesion to the mutated polypeptide. TS2/16 stimulatory ␤1-integrin antibodies or Mn 2؉ induced ␣5␤1-mediated adhesion of K562 and MV3 to GRGDSP. In the presence of TS2/16 or Mn 2؉ , ␣5␤1-mediated MV3 adhesion to the mutated polypeptide was equally strong as adhesion to the control polypeptide. Mn 2؉ or TS2/16 induced weak K562 binding to the mutated polypeptide, and in the presence of a combination of phorbol 12-myristate 13-acetate, Mn 2؉, and TS2/16, ␣5␤1-mediated K562 adhesion to the mutated and control polypeptide was equally strong. Our findings demonstrate that requirement for the PHSRN synergy site for ␣5␤1-mediated adhesion to RGD in fibronectin depends on the activation state of the integrin. Fibronectin (Fn)1 is an extracellular matrix glycoprotein that functions in cell adhesion and migration in wound healing, embryonic development, and malignant transformation (1, 2). The Fn molecule is composed of three types of repeating modules, termed type I, II, and III repeats (3), which are organized into functional domains. Proteolytic cleavage yields several fragments containing domains that promote cell adhesion, including the carboxyl-terminal HepII domain (4), the alternatively spliced type III connecting segment (5), and the central cell binding domain (CCBD).The CCBD consists of type III repeats, each containing approximately 90 amino acids (6). Cells bind to the CCBD via receptors of the integrin family (7). Integrins are ␣␤ heterodimeric transmembrane molecules mediating cell-cell adhesion and attachment of cells to the extracellular matrix (8).Integrins that bind the CCBD include ␣3␤1 (9), ␣5␤1 (10, 11), ␣v␤1 (12), ␣v␤3 (13), ␣IIb␤3 (14, 15), and ␣v␤6 (16).The Arg-Gly-Asp (RGD) sequence in the 10th type III repeat (3Fn10) is the key attachment site for binding of these integrins to the CCBD, as demonstrated by inhibition of cell adhesion with synthetic RGD-containing peptides (17, 18). Furthermore, two synergistic regions in the CCBD besides RGD have been identified that are required for cell adhesion through ␣IIb␤3 (19) and ␣5␤1 (20 -23). For ␣5␤1 binding to Fn, the synergy region in 3Fn9 is the most important of these two regions (21), and recently, a short amino acid sequence ProHis-Ser-Arg-Asn (PHSRN) was identified in this repeat that synergistically enhances the cell adhesion promoting activity of the RGD sequence (24). This sequence is also present in an 11-amin...

No abstract

The existence of XAGE genes was first reported after database homology searches for PAGE-like sequences identified 3 XAGE EST clusters. One of these clusters, XAGE-1, has in later studies been identified as a cancer/testis-associated gene. Here, we report the expression profiles of all 3 reported XAGE genes, as well as several splice variants of XAGE-1, in normal human tissues, Ewing's sarcoma and melanocytic tumors. We also provide the genetic structure of the corresponding genes. Moreover, by searching the databases for XAGE homologues, we identified 3 additional GAGE-like genes. RT-PCR studies showed frequent expression in melanoma metastases and Ewing's sarcoma for 2 XAGE-1-derived transcripts. XAGE-2 was expressed at lower frequency in these tissues, while XAGE-3 was seen only in normal placenta. Due to a frameshift, the largest XAGE-1 putative protein is far less homologous to GAGE-like proteins than the other XAGEs. Interestingly, all GAGE-like genes contain a large secondary open reading frame, coding for putative proteins homologues to the XAGE-1 primary protein. The XAGE family of cancer/testis-associated genes is located on chromosome Xp11.21-Xp11.22. The data outline a superfamily of GAGE-like cancer/testis antigens, consisting of at least 19 genes.

Suppression subtractive hybridization, comparing mRNA expression profiles of common nevocellular nevi and melanoma metastases, was used to identify potential markers of melanoma progression. From the metastases we isolated XAGE-1b, a 470 bp transcript of the XAGE-1 gene. In general, expression of XAGE-1b was much more prominent than expression of the longer XAGE-1 transcript, isolated from Ewing's sarcoma. The XAGE-1b open-reading frame codes for a putative protein of 81 amino acids, harboring a functional bipartite nuclear localization signal and a C-terminal acidic transcription-activation-like domain. On the nucleotide level, XAGE-1b has a 50% homology with members of the GAGE family. However, homology between the corresponding proteins is weak. Expression of XAGE-1b in normal tissues was mainly restricted to testis, while placenta and brain were sporadically positive. In human tumor cell lines as well as in human tumor lesions, expression was most frequently found in melanocytic tumors and Ewing's sarcoma. In the different stages of melanocytic tumor progression, expression was exclusively seen in melanoma metastases (38%; n = 61), while all tested common and atypical nevi (n = 10) as well as primary melanomas (n = 8) were negative. Upregulation of expression after treatment with demethylating agent 5-aza-2'-deoxycytidine was detected in 1 of 4 human melanoma cell lines tested. The XAGE-1 gene consists of 4 exons and is located on chromosome Xp11.21-Xp11.22. After transfection into COS cells, the corresponding protein can direct the coupled fluorescent protein to the nucleus, showing a distinct speckled staining aspect. Our data imply the nuclear cancer/testis-associated XAGE-1b to be a marker for late melanocytic tumor progression.

No abstract