The Role of the Cytoplasmic Domain of α6 Integrin in the Assembly and Function of α6β1 and α6β4

Abstract

We have studied the role of the cytoplasmic domain of a6 in the assembly and function of the a6/j'4 integrin, and compared it with the role of a6 in the assembly and function of a6/jl, by transfection of cDNAs encoding cytoplasmic mutants of (16 into K562 cells with or without full-length p4 cDNA. Des-(1 022-1050)-a6, which contains a deletion C-terminal to the GFFKR motif, was expressed in association with PI, but associated preferentially with p4, whereas the wild-type a6 subunit associated efficiently with pl and 114. Des-(1016-1050)-a6, which lacked also the GFFKR sequence, was only expressed at the cell surface when p4 was available. Transient expression in COS-7 cells showed that des-(1016-1OSO)-cx6 was retained in the endoplasmic reticulum as a monomer, which suggests that truncation of the cytoplasmic domain reduces the affinity of a6 for 81, particularly when the GFFKR sequence is absent.Although the GFFKR motif is not essential for association of a6 with p4, it increases the stability of the a6P4 integrin. The cytoplasmic domain of a6 is essential for inside-out and outside-in signaling via the a6p1 receptor, but not for adhesion via a6P4. We show that a6p4 is a constitutively active receptor. Thus, unlike adhesion by most other integrins, adhesion by a6p4 does not seem to depend on any active cellular process. Binding of u6p4 to ligand was only slightly affected by truncation of the a6 cytoplasmic domain N-terminal to the GFFKR sequence and became partially dependent on metabolic energy. These data indicate that truncations of the cytoplasmic domain of the a6 subunit affect the assembly and function of a6pl more strongly than those of a6p4. This difference may be due to the greater affinity of a6 for p4 than for 81, which makes m6P4 less susceptible to the effect of truncations.

Functional Characterization of Integrin α6β4 Adhesion Interactions Using Soluble Integrin Constructs Reveals the Involvement of Different Functional Domains in the β4 Subunit

“…Our experience supports a role for the Fc moiety in facilitating dimerization, as suggested previously for a4b1-Fc (Stephens et al 2000). Furthermore, subunit cytoplasmic and transmembrane regions are known to play a role in heterodimerization and surface expression of many integrins, including a6b4 (Briesewitz et al 1993;De Melker and Sonnenberg 1996;Lu and Springer 1997), suggesting addition of the Fc in this case was necessary for successful production of the a6b4 ectodomain soluble proteins. Both a6-Fcb4 and a6b4-Fc proteins exhibited similar behavior patterns as endogenously expressed a6b4 in terms of ligand and ion dependency of adhesion interactions, as well as recognition by a6-or b4-specific antibodies.…”

Expression of a Soluble Functional Form of the Integrinα4β1in Mammalian Cells

“…The cytoplasmic and transmembrane regions of integrins have been reported to play a role in heterodimer formation (Pardi et al, 1995;Briesewitz et al, 1993;De Melker and Sonnenberg, 1996;Lu and Springer, 1997). These regions are normally deleted to generate soluble integrins.…”

The two phenylalanines in the GFFKR motif of the integrin α6A subunit are essential for heterodimerization

“…In contrast, α2, α4 and α6 subunits truncated N-terminal of the GFFKR motif did not reach the cell surface [17][18][19], and the levels of similar mutants of α5 [20] and αL [21] at the cell surface were very low. This suggests that the GFFKR motif is important for subunit association of integrins and their transport to the plasma membrane.…”