H. Kashiwagi et al., A mutation in the extracellular cysteine-rich repeat region of the beta(3)subunit activates integrins alpha(IIb)beta(3) and alpha(V)beta(3), BLOOD, 93(8), 1999, pp. 2559-2568
Inside-out signaling regulates the ligand-binding function of integrins thr
ough changes in receptor affinity and/or avidity. For example, alpha(IIb)be
ta(3) is in a low-affinity/avidity state in resting platelets, and activati
on of the receptor by platelet agonists enables fibrinogen to bind. In addi
tion, certain mutations and truncations of the integrin cytoplasmic tails a
re associated with a high-affinity/avidity receptor. To further evaluate th
e structural basis of integrin activation, stable Chinese hamster ovary (CH
O) cell transfectants were screened for high-affinity/avidity variants of a
lpha(IIb)beta(3) One clone (AM-1) expressed constitutively active alpha(IIb
)beta(3), as evidenced by (1) binding of soluble fibrinogen and PAC1, a lig
and-mimetic alpha(IIb)beta(3) antibody; and (2) fibrinogen-dependent cell a
ggregation. Sequence analysis and mutant expression in 293 cells proved tha
t a single amino acid substitution in the cysteine-rich, extracellular port
ion of beta(3)(T562N) was responsible for receptor activation. In fact, T56
2N also activated alpha(v)beta(3), leading to spontaneous binding of solubl
e fibrinogen to 293 cells. In contrast, neither T562A nor T562Q activated a
lpha(IIb)beta(3) suggesting that acquisition of asparagine at residue 562 w
as the relevant variable. T562N also led to aberrant glycosylation of beta(
3), but this was not responsible for the receptor activation. The binding o
f soluble fibrinogen to alpha(IIb)beta(3)(T562N) was not sufficient to trig
ger tyrosine phosphorylation of pp125(FAK), indicating that additional post
-ligand binding events are required to activate this protein tyrosine kinas
e during integrin signaling. These studies have uncovered a novel gain-of-f
unction mutation in a region of beta(3) intermediate between the ligand-bin
ding region and the cytoplasmic tail, and they suggest that this region is
involved in integrin structural changes during inside-out signaling. (C) 19
99 by The American Society of Hematology.