Abstract
Integrin transmembrane (TM) and/or cytoplasmic domains play a critical role in integrin bidirectional signaling. Although it has been shown that TM and/or cytoplasmic α and β domains associate in the resting state and separation of these domains is required for both inside‐out and outside‐in signaling, the role of TM homomeric association remains elusive. Formation of TM homo‐oligomers was observed in micelles and bacterial membranes previously, and it has been proposed that homomeric association is important for integrin activation and clustering. This study addresses whether integrin TM domains form homo‐oligomers in mammalian cell membranes using cysteine scanning mutagenesis. Our results show that TM homomeric interaction does not occur before or after soluble ligand binding, or during inside‐out activation. In addition, even though the cysteine mutants and the heterodimeric disulfide‐bounded mutant could form clusters after adhering to immobilized ligand, the integrin TM domains do not form homo‐oligomers, suggesting that integrin TM homomeric association is not critical for integrin clustering or outside‐in signaling. Therefore, integrin TM homo‐oligomerization is not required for integrin activation, ligand binding or signaling.
Highlights
None of the ␣IIb or 3 TM cysteine mutations formed a homomeric disulfide bond (Figs. 6 and supplemental Fig. 3). Because these integrins adhered to the immobilized fibrinogen, clustered on the cell surface and transmitted outside-in signaling leading to cell spreading, the results suggest that homomeric association of the integrin TM domains is not important for integrin functions
It has been shown that TM heterodimeric association stabilizes integrins in the resting state; when integrins are activated by intracellular signals, two TM/cytoplasmic tails separate, leading to conformational change of the ligand binding extracellular regions, resulting in high affinity for ligands (5, 8, 9, 12, 13, 16 –18, 45)
Based on the observation that integrin TM helices formed homo-oligomers using recombinant peptides in detergent [23, 29, 31], the GALLEX assay in bacteria [45], the TOXCAT assay in bacteria [24, 25], and the computational modeling (26 –28), TM homomeric association has been suggested to be important for integrin activation [14, 29, 31]
Summary
Recommended Citation Wang, W., Zhu, J., Springer, T., & Luo, B. (2011). Tests of integrin transmembrane domain homooligomerization during integrin ligand binding and signaling. Inside-out activation occurs when specific intracellular molecules such as talin and kindlin bind to the integrin cytoplasmic domain, leading to integrin conformational change and high affinity for extracellular ligands. It was proposed that integrin TM homo-oligomerization is not a critical step for inside-out activation, but instead, it may help to stabilize the integrin in the high affinity state and be important for outside- in signaling [24]. The mechanism of how integrins transmit these signals across the plasma membrane through the TM/ cytoplasmic domains remains unknown It is unclear whether integrin TM homo-oligomerization plays any role in integrin clustering and signaling, even though it has been proposed that it provides structural basis for this process [29, 30]. Our results show that integrin TM domains do not form homo-oligomers before or after soluble ligand binding or during integrin inside-out and outside-in signaling
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