A genetic analysis of integrin function: Glanzmann thrombasthenia in vitro.
Glanzmann thrombasthenia, an inherited bleeding disorder, can be caused by a defect or deficiency in platelet integrin alphaIIb beta3 (GPIIb-IIIa). Studies of thrombasthenia variants have facilitated identification of sites involved in the functions of alphaIIb beta3 and other integrins. Such sites include those that bind ligand and those that participate in the "activation" of alphaIIb beta3 required for high affinity binding of ligands such as fibrinogen or PAC1, a monoclonal antibody. Here we describe the isolation of such variants, created in vitro with Chinese hamster ovary cells that express an activated form of alphaIIb beta3. These cells were exposed to a mutagen, ethyl methane sulfonate, and variants that lost the capacity to bind PAC1 were isolated by fluorescence-activated cell sorting. These variants were grouped into three phenotypic classes. One comprised integrin mutations that disrupt ligand binding function; a second comprised mutations that interfere with the capacity of cells to activate the integrin. Most of these activation-defective mutations were in the integrin cytoplasmic domain, but surprisingly, several were caused by mutations affecting three closely spaced residues in the beta3 extracellular domain. A third class of mutants exhibited a defect in integrin activation not ascribable to changes in the integrin sequence. Thus, these may represent mutated signaling molecules required for integrin activation. This unbiased genetic approach provides new insights into the structural basis of integrin function and may assist in identifying the cellular events that regulate integrin function.
Baker
E K
EK
Department of Vascular Biology, Scripps Research Institute, La Jolla, CA 92037, USA.
Tozer
E C
EC
Pfaff
M
M
Shattil
S J
SJ
Loftus
J C
JC
Ginsberg
M H
MH
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D001665:Binding Sites; D001792:Blood Platelets / Q000737:chemistry / Q000378:metabolism*; D016466:CHO Cells; D002465:Cell Movement / Q000235:genetics; D006224:Cricetinae; D005020:Ethyl Methanesulfonate / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D016023:Integrins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D013915:Thrombasthenia / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D016023:Integrins; D008024:Ligands; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011994:Recombinant Proteins; D005020:Ethyl Methanesulfonate