MOLECULAR ANALYSES OF CELL-MATRLX ADHESION 
Richard O. Hynes, Ph.D., Investigator 
The laboratory is involved in molecular and 
cellular analyses of cell adhesion and its role in a 
variety of physiological processes, including em- 
bryological development, hemostasis, thrombosis, 
wound healing, and cancer. The research is concen- 
trated on a set of adhesive extracellular matrix 
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 of which are derived from a single 
gene by alternative splicing of the initial 70 kb tran- 
script to give multiple mRNAs of 8-9 kb. These 
mRNAs differ by inclusion or exclusion of three seg- 
ments and therefore encode slightly different pro- 
teins. One area of research concerns the molecular 
basis and physiological consequences of the alter- 
native splicing of fibronectins. The alternatively 
spliced exons are differentially expressed in differ- 
ent cells and tissues, and the pattern of splicing is 
altered during development and in response to 
physiological stimuli. For example, two of the seg- 
ments (A and B) are always present in the 
fibronectin associated with cell migration during 
development. They are selectively excluded by vari- 
ous cell types later in development; e.g., both are 
excluded from fibronectin mRNA in adult skin. 
However, after wounding of the skin there is a 
marked increase in the levels of fibronectin mRNA, 
and fibronectin contains both A and B segments, as 
in embryos. This suggests that A''^B^ fibronectin 
may be important for the migration and/or prolifer- 
ation that occurs both in developing embryos and 
in healing wounds. 
To test this hypothesis and others based on the 
descriptive studies of the expression and splicing 
patterns of different fibronectin isoforms, recombi- 
nant fibronectin genes were constructed and in- 
troduced into cells to produce cell lines that se- 
crete in pure, homogeneous form each of the forms 
of fibronectin that, in nature, are found in 
mixtures. In this way it is possible to purify the 
different forms in quantity and to assay their biolog- 
ical functions. One result of these studies is that 
certain lymphoid cells adhere specifically only to 
those forms of fibronectin that contain the third al- 
ternatively 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^p^) that recog- 
nizes this site was identified by affinity chromatogra- 
phy on synthetic peptides and by specific antibody 
blocking. 
Studies are under way to analyze further the 
roles of the three alternatively spliced segments, 
both in vitro and in vivo. Projects have been initi- 
ated to prepare transgenic mice expressing specific 
forms of fibronectin inappropriately and to "knock 
out" the fibronectin gene in embryonic stem (ES) 
cells in order to generate mice with mutant 
fibronectin genes. Finally, using DNA hybridization 
and polymerase chain reaction, attempts are in 
progress to identify and clone the gene for 
fibronectin in Drosophila. Success in this latter 
project would open the way to the application of 
the sophisticated genetic manipulations possible in 
this organism. 
Integrins are transmembrane receptors made up 
of a- and P-subunits. There are at least 6 P-subunits 
and at least 11 a-subunits. Different ap combina- 
tions generate receptors with different but over- 
lapping specificities for various adhesive extracel- 
lular matrix proteins. The a- and P-subunits 
interact via their large extracellular domains with 
these adhesive proteins and via their small cytoplas- 
mic domains with cytoskeletal 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 certain in- 
tegrins; this loss probably contributes to their 
altered ability to adhere to and assemble extracellu- 
lar matrices. This model is under test by trans- 
fection experiments. 
The roles of integrins in development are being 
studied genetically in Drosophila. The gene for a 
Drosophila P-integrin has been cloned, and mu- 
tants are available. Gynandromorphs and somatic 
clones reveal specific defects in patches where in- 
tegrins are deleted. Reintroduction of wild-type and 
mutant integrins into the mutant strains will allow 
further analysis of the roles of these proteins. 
The nature of the cytoskeletal connection is 
being examined by transfection of mutated integrin 
subunit genes into cells. These experiments impli- 
cate the cytoplasmic domain of the P^-subunit in as- 
sociation with the cytoskeleton. Similar studies are 
being initiated on the a-subunits, with particular at- 
tention to three related integrin receptors («^Pj, 
Continued 
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