Retroviral expression of alternatively spliced forms of rat fibronectin.
We describe the construction in retroviral vectors and the expression of recombinant rat fibronectin (FN) cDNAs corresponding with the various alternatively spliced forms of FN. In NIH 3T3 cells, the exogenous rat FN subunits are efficiently secreted as heterodimers with endogenous mouse subunits. In contrast, in lymphoid WEHI231 cells, there is no endogenous FN synthesis and the recombinant FNs are secreted and can be purified as homogeneous proteins. We show that the purified recombinant FNs are biochemically and biologically functional. In basic assays for adhesion, spreading, cytoskeletal organization, and migration using various established adherent cell lines, different forms of FNs containing the different alternatively spliced segments show no marked differences in activity. We have used these recombinant FNs to investigate three systems in which earlier results had suggested potential differences between different forms of FN. First, all forms tested appear equally active in restoring normal morphology to a transformed cell line. Second, we detect minor differences in their ability to assemble into preexisting extracellular matrices. Finally, we report that only those forms of FN that contain the V segment will promote the spreading of a lymphoid cell line indicating that this segment confers additional biological functions for some cell types, a result that confirms and extends earlier data. These homogeneous, biologically active recombinant FNs will allow further studies of the role of the alternatively spliced segments of FN.
Guan
J L
JL
Cancer for Cancer Research, Massachusetts Institute of Technology, Cambridge 02139.
Trevithick
J E
JE
Hynes
R O
RO
D000818:Animals; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics; D005353:Fibronectins / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D015870:Gene Expression; D005796:Genes; D005822:Genetic Vectors; D046911:Macromolecular Substances; D008546:Melanoma, Experimental; D051379:Mice; D010957:Plasmids; D012326:RNA Splicing; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000494:pharmacology; D012190:Retroviridae / Q000235:genetics*; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000187:drug effects
D005353:Fibronectins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D004247:DNA