Alexander R. Kazarov

An extracellular site on tetraspanin CD151 determines α3 and α6 integrin–dependent cellular morphology

The α3β1 integrin shows strong, stoichiometric, direct lateral association with the tetraspanin CD151. As shown here, an extracellular CD151 site (QRD194–196) is required for strong (i.e., Triton X-100–resistant) α3β1 association and for maintenance of a key CD151 epitope (defined by monoclonal antibody TS151r) that is blocked upon α3 integrin association. Strong CD151 association with integrin α6β1 also required the QRD194–196 site and masked the TS151r epitope. For both α3 and α6 integrins, strong QRD/TS151r-dependent CD151 association occurred early in biosynthesis and involved α subunit precursor forms. In contrast, weaker associations of CD151 with itself, integrins, or other tetraspanins (Triton X-100–sensitive but Brij 96–resistant) were independent of the QRD/TS151r site, occurred late in biosynthesis, and involved mature integrin subunits. Presence of the CD151–QRD194–196→INF mutant disrupted α3 and α6 integrin–dependent formation of a network of cellular cables by Cos7 or NIH3T3 cells on basement membrane Matrigel and markedly altered cell spreading. These results provide definitive evidence that strong lateral CD151–integrin association is functionally important, identify CD151 as a key player during α3 and α6 integrin–dependent matrix remodeling and cell spreading, and support a model of CD151 as a transmembrane linker between extracellular integrin domains and intracellular cytoskeleton/signaling molecules.

Tetraspanin CD151 regulates alpha6beta1 integrin adhesion strengthening.

The tetraspanin CD151 molecule associates specifically with laminin-binding integrins, including α6β1. To probe strength of α6β1-dependent adhesion to laminin-1, defined forces (0–1.5 nN) were applied to magnetic laminin-coated microbeads bound to NIH 3T3 cells. For NIH 3T3 cells bearing wild-type CD151, adhesion strengthening was observed, as bead detachment became more difficult over time. In contrast, mutant CD151 (with the C-terminal region replaced) showed impaired adhesion strengthening. Static cell adhesion to laminin-1, and detachment of beads coated with fibronectin or anti-α6 antibody were all unaffected by CD151 mutation. Hence, CD151 plays a key role in selectively strengthening α6β1 integrin-mediated adhesion to laminin-1.

Tetraspanin CD151 regulates α6β1 integrin adhesion strengthening

The tetraspanin CD151 molecule associates specifically with laminin-binding integrins, including α6β1. To probe strength of α6β1-dependent adhesion to laminin-1, defined forces (0–1.5 nN) were applied to magnetic laminin-coated microbeads bound to NIH 3T3 cells. For NIH 3T3 cells bearing wild-type CD151, adhesion strengthening was observed, as bead detachment became more difficult over time. In contrast, mutant CD151 (with the C-terminal region replaced) showed impaired adhesion strengthening. Static cell adhesion to laminin-1, and detachment of beads coated with fibronectin or anti-α6 antibody were all unaffected by CD151 mutation. Hence, CD151 plays a key role in selectively strengthening α6β1 integrin-mediated adhesion to laminin-1.

Diminished metastasis in tetraspanin CD151-knockout mice

Tetraspanin protein CD151 on tumor cells supports invasion and metastasis. In the present study, we show that host animal CD151 also plays a critical role. CD151-null mice showed markedly diminished experimental lung metastasis after injection of Lewis lung carcinoma or B16F10 melanoma cells. Diminished tumor cell residence in the lungs was evident 6-24 hours after injection. Consistent with an endothelial cell deficiency, isolated CD151-null mouse lung endothelial cells showed diminished support for B16F10 adhesion and transendothelial migration, diminished B16F10-induced permeability, and diminished B16F10 adhesion to extracellular matrix deposited by CD151-null mouse lung endothelial cells. However, CD151 deletion did not affect the size of metastatic foci or subcutaneous primary B16F10 tumors, tumor aggregation, tumor clearance from the blood, or tumor-induced immune cell activation and recruitment. Therefore, the effects of host CD151 on metastasis do not involve altered local tumor growth or immune surveillance. VEGF-induced endothelial cell signaling through Src and Akt was diminished in CD151-null endothelial cells. However, deficient signaling was not accompanied by reduced endothelial permeability either in vitro (monolayer permeability assay) or in vivo (VEGF-stimulated Miles assay). In summary, diminished metastasis in CD151-null host animals may be due to impaired tumor-endothelial interactions, with underlying defects in mouse lung endothelial cell extracellular matrix production.

Tetraspanin CD151 regulates 6 1 integrin adhesion strengthening

The tetraspanin CD151 molecule associates specifically with laminin-binding integrins, including α6β1. To probe strength of α6β1-dependent adhesion to laminin-1, defined forces (0–1.5 nN) were applied to magnetic laminin-coated microbeads bound to NIH 3T3 cells. For NIH 3T3 cells bearing wild-type CD151, adhesion strengthening was observed, as bead detachment became more difficult over time. In contrast, mutant CD151 (with the C-terminal region replaced) showed impaired adhesion strengthening. Static cell adhesion to laminin-1, and detachment of beads coated with fibronectin or anti-α6 antibody were all unaffected by CD151 mutation. Hence, CD151 plays a key role in selectively strengthening α6β1 integrin-mediated adhesion to laminin-1.