The integrin alpha IIb-beta 3, platelet glycoprotein IIb-IIIa, can form a functionally active heterodimer complex without the cysteine-rich repeats of the beta 3 subunit.
Integrin alpha IIb-beta 3 binds fibrinogen via the recognition sequence Arg-Gly-Asp-Ser (RGDS). We have used the baculovirus/insect cell expression system to study the structural requirements for the formation of a functionally active fragment of alpha IIb-beta 3. A tandem baculovirus transfer vector was constructed containing the cDNA coding for the heavy chain of human alpha IIb (alpha IIbH, amino acids 1-874) and the cDNA coding for a truncated form of human beta 3 (t beta 3; amino acids 1-469). Sf9 insect cells were infected with the corresponding baculovirus, and the produced soluble recombinant proteins were purified using an RGD-like affinity column. The bound receptor fragments were specifically eluted with RGDS and existed as a heterodimeric complex (rec alpha IIbH-t beta 3) with an apparent M(r) of 160,000. In an immunocapture assay, the monoclonal antibody pl-55, which only recognizes the functionally active form of alpha IIb-beta 3, bound to the purified complex. Rec alpha IIbH-t beta 3 specifically bound 125I-fibrinogen with an affinity comparable with that of purified platelet alpha IIb-beta 3. Electron micrographs of rotary-shadowed rec alpha IIbH-t beta 3 showed that the complex had the characteristic globular head, but the two rodlike tails were 4-6 nm shorter than those found in intact alpha IIb-beta 3. Thus, the cysteine-rich repeats of beta 3 are not required for assembly, stability, and functional activity of this integrin.
Wippler
J
J
Pharma Division, F. Hoffmann-La Roche Ltd., Basel, Switzerland.
Kouns
W C
WC
Schlaeger
E J
EJ
Kuhn
H
H
Hadvary
P
P
Steiner
B
B
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D002413:Cations, Divalent; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D016023:Integrins / Q000737:chemistry* / Q000378:metabolism; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins; D012091:Repetitive Sequences, Nucleic Acid; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D002413:Cations, Divalent; D016023:Integrins; D010980:Platelet Membrane Glycoproteins; D011994:Recombinant Proteins; D005340:Fibrinogen; D003545:Cysteine