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ICAP-1, a novel beta1 integrin cytoplasmic domain-associated protein, binds to a conserved and functionally important NPXY sequence motif of beta1 integrin.

ABSTRACT: The cytoplasmic domains of integrins are essential for cell adhesion. We report identification of a novel protein, ICAP-1 (integrin cytoplasmic domain- associated protein-1), which binds to the 1 integrin cytoplasmic domain. The interaction between ICAP-1 and beta1 integrins is highly specific, as demonstrated by the lack of interaction between ICAP-1 and the cytoplasmic domains of other beta integrins, and requires a conserved and functionally important NPXY sequence motif found in the COOH-terminal region of the beta1 integrin cytoplasmic domain. Mutational studies reveal that Asn and Tyr of the NPXY motif and a Val residue located NH2-terminal to this motif are critical for the ICAP-1 binding. Two isoforms of ICAP-1, a 200-amino acid protein (ICAP-1alpha) and a shorter 150-amino acid protein (ICAP-1beta), derived from alternatively spliced mRNA, are expressed in most cells. ICAP-1alpha is a phosphoprotein and the extent of its phosphorylation is regulated by the cell-matrix interaction. First, an enhancement of ICAP-1alpha phosphorylation is observed when cells were plated on fibronectin-coated but not on nonspecific poly-L-lysine-coated surface. Second, the expression of a constitutively activated RhoA protein that disrupts the cell-matrix interaction results in dephosphorylation of ICAP-1alpha. The regulation of ICAP-1alpha phosphorylation by the cell-matrix interaction suggests an important role of ICAP-1 during integrin-dependent cell adhesion.

SUBMITTER: Chang DD

PROVIDER: S-EPMC2136751 | biostudies-literature | 1997 Sep

REPOSITORIES: biostudies-literature

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ICAP-1, a novel beta1 integrin cytoplasmic domain-associated protein, binds to a conserved and functionally important NPXY sequence motif of beta1 integrin.

Chang D D DD Wong C C Smith H H Liu J J

The Journal of cell biology 19970901 5

The cytoplasmic domains of integrins are essential for cell adhesion. We report identification of a novel protein, ICAP-1 (integrin cytoplasmic domain- associated protein-1), which binds to the 1 integrin cytoplasmic domain. The interaction between ICAP-1 and beta1 integrins is highly specific, as demonstrated by the lack of interaction between ICAP-1 and the cytoplasmic domains of other beta integrins, and requires a conserved and functionally important NPXY sequence motif found in the COOH-ter ...[more]

PMID: 9281591

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Project description:Protein interactions with the integrin beta-subunit cytoplasmic domain (beta-tail) are essential for adhesion-dependent processes, including cell spreading and the connection of integrins with actin filaments at adhesion sites. Talin-1 binds to the conserved membrane-proximal NPxY motif of beta-tails (NPIY in beta1 integrin) promoting the inside-out activation of integrins and providing a linkage between integrins and the actin cytoskeleton. Here, we characterize the role of interactions between talin-1 and beta-tail downstream of integrin activation, in the context of recombinant integrins containing either the wild type (WT) or the (YA) mutant beta1A tail, with a tyrosine to alanine substitution in the NPIY motif. In addition to inhibiting integrin activation, the YA mutation suppresses cell spreading, integrin signaling, focal adhesion and stress-fiber formation, as well as microtubule assembly. Constitutive activation of the mutant integrin restores these integrin-dependent processes, bringing into question the importance of the NPIY motif downstream of integrin activation. Depletion of talin-1 using TLN1 siRNA demonstrated that talin-1 is required for cell spreading, focal adhesion and stress-fiber formation, as well as microtubule assembly, even when cells are adhered by constitutively activated WT integrins. Depletion of talin-1 does not inhibit these processes when cells are adhered by constitutively activated mutant integrins, suggesting that the binding of an inhibitory protein to the NPIY motif negatively regulates integrin function when talin-1 is depleted. We identified filamin A (FLNa) as this inhibitory protein; it binds to the beta1A tail in an NPIY-dependent manner and inhibition of FLNa expression in talin-1-depleted cells restores integrin function when cells are adhered by constitutively activated WT integrins. FLNa binds FilGAP, which is a negative regulator of Rac activation. Expression of the dominant inhibitory mutant, FilGAP(DeltaGAP), which lacks GAP activity restores spreading in cells adhered by constitutively activated integrins containing the beta1A tail, but not by integrins containing the beta1D tail, which is known to bind poorly to FLNa. Together, these results suggest that the binding of talin-1 to the NPIY motif is required downstream of integrin activation to promote cell spreading by preventing the inappropriate recruitment of FLNa and FilGAP to the beta1A tail. Our studies emphasize the importance of understanding the mechanisms that regulate the differential binding FLNa and talin-1 to the beta1 tail downstream of integrin activation in promoting integrin function.

Dataset Information

ICAP-1, a novel beta1 integrin cytoplasmic domain-associated protein, binds to a conserved and functionally important NPXY sequence motif of beta1 integrin.

Publications

ICAP-1, a novel beta1 integrin cytoplasmic domain-associated protein, binds to a conserved and functionally important NPXY sequence motif of beta1 integrin.

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