Analysis of the CD151·α3β1 Integrin and CD151·Tetraspanin Interactions by Mutagenesis

Abstract

Transmembrane proteins of the tetraspanin superfamily are associated with various integrins and modulate their function. We performed mutagenesis analysis to establish structural requirements for the interaction of CD151 with the ␣ 3 ␤ 1 integrin and with other tetraspanins. Using a panel of CD151/CD9 chimeras and CD151 deletion mutants we show that the minimal region, which confers stable (e.g. Triton X-100-resistant) association of the tetraspanin with ␣ 3 ␤ 1 , maps within the large extracellular loop (LECL) of CD151 (the amino acid sequence between residues Leu 149 and Glu 213 ). Furthermore, the substitution of 11 amino acids (residues 195-205) from this region for a corresponding sequence from CD9 LECL or point mutations of cysteines in the conserved CCG and PXXCC motifs abolish the interaction. The removal of the LECL CD151 does not affect the association of the protein with other tetraspanins (e.g. CD9, CD81, CD63, and wild-type CD151). On the other hand, the mutation of the CCG motif selectively prevents the homotypic CD151⅐CD151 interaction but does not influence the association of the mutagenized CD151 with other tetraspanins. These results demonstrate the differences in structural requirements for the heterotypic and homotypic tetraspanin⅐tetraspanin interactions. Various deletions involving the small extracellular loop and the first three transmembrane domains prevent surface expression of the CD151 mutants but do not affect the CD151⅐␣ 3 ␤ 1 interaction. The CD151 deletion mutants are accumulated in the endoplasmic reticulum and redirected to the lysosomes. The assembly of the CD151⅐␣ 3 ␤ 1 complex occurs early during the integrin biosynthesis and precedes the interaction of CD151 with other tetraspanins. Collectively, these data show that the incorporation of CD151 into the "tetraspanin web" can be controlled at various levels by different regions of the protein.

Tetraspanin CD151 regulates α6β1 integrin adhesion strengthening

“…Our data now support a ''transmembrane linker'' model in which (i) the integrin contacts laminin, (ii) CD151 forms an extracellular contact with the ␣ subunit of laminin-binding integrins (31,35), and (iii) CD151 uses its short cytoplasmic tail to engage as yet unidentified membraneproximal elements. These unknown elements are critical for strengthening integrin-mediated adhesion, such that it withstands larger mechanical forces.…”

The Ig Domain Protein CD9P-1 Down-regulates CD81 Ability to Support Plasmodium yoelii Infection

“…The best characterized tetraspanin/partner pair is CD151/integrin ␣3␤1, and several studies have revealed the importance of the CD151 large extracellular domain for this interaction (37,38). This is consistent with the LEL of tetraspanins being the most variable region of these molecules (14 -16).…”

Clarin-1, Encoded by the Usher Syndrome III Causative Gene, Forms a Membranous Microdomain

“…Unlike tetraspanin, CLRN1 does not conserve the structurally essential cysteine residues in the second extracellular loop that tetraspanins dedicate to forming intramolecular disulfide-bonds. These disulfide bonds were found to be required for the heterophilic interaction of tetraspanin with its cognate integrin, as well as for the homophilic interaction between tetraspanins themselves (47). Although CLRN1 has no cysteine residues in the second extracellular loop, significant interactions between CLRN1 and integrin subunits still occur.…”