A naturally occurring point mutation in the beta3 integrin MIDAS-like domain affects differently alphavbeta3 and alphaIIIbbeta3 receptor function.
We have investigated the effect of a new Leu196Pro mutation, identified in the MIDAS-like domain of the beta3 integrin subunit in a patient with type II Glanzmann thrombasthenia, on beta3 integrin receptor function. Expression of the mutant beta3Pro196 subunit in CHO cells, either associated with recombinant human alphaIIb or alphav, resulted in normal biosynthesis of beta3 and heterodimerization with alphav or alphaIIb, but selectively interfered with alphaIIbbeta3 maturation and transport to the cell surface. Functional analysis of the beta3 mutant receptors revealed strong inhibition of alphavbeta3-mediated cell spreading on immobilized fibrinogen, focal contact formation, p125FAK phosphorylation and fibrin clot retraction, as opposed to normal alphaIIbbeta3-mediated cell interaction with immobilized fibrinogen, focal contact translocation and signaling. In contrast, antibody- or DTT-activated mutant aIIbbeta3 was unable to bind soluble fibrinogen or the ligand mimetic PAC-1 monoclonal antibody, but underwent a conformational change following RGD peptide binding as demonstrated by AP5-LIBS epitope expression. These results suggest that (1) the highly conserved TL196T motif in the beta3 integrin subunit is located in a domain structurally important for the exposure of a functional binding site for soluble fibrinogen; and (2) that the MIDAS-like contact site in beta3 is not involved in alphaIIbbeta3-mediated cell adhesion to immobilized fibrinogen, while it is essential for alphavbeta3-mediated interaction with this ligand.
Morel-Kopp
M C
MC
Laboratoire Franco-Luxembourgeois de Recherche Biomédicale (CNRS/CRP-Santé), Centre Universitaire, Luxembourg.
Melchior
C
C
Chen
P
P
Ammerlaan
W
W
Lecompte
T
T
Kaplan
C
C
Kieffer
N
N
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015703:Antigens, CD / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000502:physiology; D016466:CHO Cells; D003018:Clot Retraction; D003062:Codon / Q000235:genetics; D006224:Cricetinae; D003412:Cricetulus; D019281:Dimerization; D005260:Female; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D039661:Integrin beta3; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D010973:Platelet Adhesiveness / Q000235:genetics / Q000502:physiology; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000235:genetics* / Q000502:physiology; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000502:physiology; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D019030:Receptors, Vitronectin / Q000235:genetics* / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000502:physiology; D013329:Structure-Activity Relationship; D013915:Thrombasthenia / Q000235:genetics*
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D003062:Codon; D039661:Integrin beta3; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D010980:Platelet Membrane Glycoproteins; D019030:Receptors, Vitronectin; D011993:Recombinant Fusion Proteins; D005340:Fibrinogen