MOLECULAR ANALYSES OF CELL-MATRIX ADHESION 
Richard O. Hynes, Ph.D., Investigator 
Dr. Hynes and his colleagues are involved in mo- 
lecular and cellular analyses of cell adhesion and its 
role in a variety of physiological processes, includ- 
ing embryological development, hemostasis, throm- 
bosis, wound healing, and cancer. The research is 
concentrated on a set of adhesive extracellular ma- 
trix proteins known as fibronectins and on a set of 
cell surface receptors known as integrins. 
Fibronectins comprise a set of related but differ- 
ent proteins, all derived from a single gene by alter- 
native splicing of the initial 70-kb transcript to give 
multiple mRNAs of 8-9 kb. These mRNAs differ by 
inclusion or exclusion of three segments and there- 
fore encode slightly different proteins. One area of 
research concerns the molecular basis and the physi- 
ological consequences of the alternative splicing of 
fibronectins. The alternatively spliced exons are dif- 
ferentially expressed in different cells and tissues, 
and the pattern of splicing is altered during develop- 
ment and in response to physiological stimuli. Two 
of the segments (A and B) are always present in the 
fibronectin associated with cell migration during de- 
velopment but are selectively excluded by various 
cell types later in development; e.g., both are ex- 
cluded from fibronectin mRNA in adult skin. How- 
ever, after wounding of the skin there is a marked 
increase in the levels of fibronectin mRNA, and this 
mRNA contains both A and B segments, as in em- 
bryos. This suggests that A^B^ fibronectin may be 
important for the migration and/or proliferation 
that occurs both in developing embryos and in heal- 
ing wounds. 
To test this and other hypotheses based on studies 
of the patterns of expression of different fibronectin 
isoforms. Dr. Hynes and his colleagues constructed 
recombinant fibronectin genes and introduced 
them into cells. Cell lines were produced that se- 
crete in pure, homogeneous form each of the types 
of fibronectin that, in nature, are found in mixtures. 
In this way it is possible to purify the different types 
in quantity and to assay their biological functions. 
One result arising from these studies is that certain 
lymphoid cells adhere specifically only to those 
forms of fibronectin containing the third alterna- 
tively spliced segment (V). The binding site within 
the V region was mapped to a 10-amino acid 
stretch. These results define an alternatively spliced 
cell-type-specific cell adhesion site in fibronectin. 
The integrin receptor (a^l3^) recognizing this site 
was identified by affinity chromatography on syn- 
thetic peptides and by specific antibody blocking. 
Similar studies are under way to analyze further the 
roles of the other alternatively spliced segments in 
the behavior of various cell types. 
To investigate the roles of different fibronectin 
isoforms in vivo, Dr. Hynes and his colleagues are 
altering the expression of fibronectins in strains of 
mice. Homologous recombination in embryonic 
stem (ES) cells is being used to "knock out" the 
fibronectin gene or to mutate it to alter its pattern of 
splicing. The altered ES cells have been reintro- 
duced into embryos, and mutant strains of mice have 
been derived. These strains reveal that fibronectin is 
essential for early embryonic development, since 
the embryos die early in development. Further analy- 
ses are under way to elucidate the exact defects pro- 
duced by elimination or alterations of the fibronec- 
tin gene. Transgenic mice expressing specific 
isoforms of fibronectin have also been generated 
and are currently being analyzed. Interbreeding of 
these various strains of mice will allow detailed ge- 
netic analyses of the roles of the different forms in 
development and in various physiological and 
pathological processes. 
The second major area of interest in the laboratory 
concerns integrins, transmembrane receptors made 
up of a and 13 subunits. There are at least 8 /3 sub- 
units and at least 12 a subunits. Different combi- 
nations generate receptors with different but 
overlapping specificities for various adhesive extra- 
cellular matrix proteins, including fibronectins. 
The a and subunits interact, via their large extra- 
cellular domains, with these adhesive proteins and, 
via their small cytoplasmic domains, with cytoskele- 
tal proteins. Thus they serve to link the extracellular 
matrix to the cytoskeleton. The spectra of integrins 
expressed by different cells vary and alter during 
development and in response to various stimuli. For 
example, oncogenically transformed cells lose cer- 
tain integrins; this loss probably contributes to their 
altered ability to adhere to and assemble extracellu- 
lar matrices and thus to their aberrant behavior. This 
model is under test by transfection experiments, in 
which integrin genes are introduced into cells and 
their properties analyzed. 
The roles of several integrins in vivo are being 
studied by homologous recombination in murine ES 
cells and generation of mutant mice, as described 
above for fibronectins. The roles of integrins in de- 
velopment are also being studied genetically in the 
fruit fly Drosophila. The genes for several Drosoph- 
ila integrin subunits have been cloned, and mutants 
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