Figure 1
Figure 1. A model for integrin activation regulated by the membrane-proximal cytoplasmic salt bridge and generation of α4-R/AGFFKR mice in which the cytoplasmic salt bridge is disrupted. (A) A positively charged RGFFKR (a circled plus) forms the membrane-proximal salt bridge with a negatively charged residue(s) (a circled minus) in the β-subunit. The salt bridge facilitates clasping of the α/β cytoplasmic domains, maintaining the extracellular domains in a default inactive conformation (left). Disruption of the salt bridge, which is induced via binding of cytoplasmic proteins such as talin (not shown), triggers unclasping of the cytoplasmic domains, converting the extracellular domains to the active conformation (right). The α- and β-subunits are labeled in the extracellular domains. The plasma membrane is shown with 2 parallel dashed lines. (B) Amino acid sequence alignment of the membrane-proximal cytoplasmic domains of selected integrin α-subunits. A conserved GFFKR motif is highlighted. The arginine residues in the GFFKR motif (RGFFKR) are shown in bold. (C) Targeted insertion of the floxed ACN cassette containing both the neor gene and the Cre-recombinase gene under the control of the sperm-specific ACE promoter,25 mutated exon 28 (28**), and UTR into the Itga4 locus, followed by deletion of the ACN cassette. The configurations of the targeting vector, α4 integrin genomic locus, the targeted allele following homologous recombination, and the ACN-deleted allele are shown. Exons are displayed as black boxes and the floxed ACN cassette, UTR, and diphtheria toxin cassette (DT) are displayed as white boxes. loxP sites are displayed as triangles. The external probe containing exon 24, which was used to verify the targeting event, is indicated. The map displays the following restriction sites: E indicates EcoRI; S, ScaI; Sa, SacI; X, XbaI; and X*, engineered XbaI. (D) Genotyping and confirmation of the deleted ACN cassette by PCR. PCR bands are shown for wild-type (WT/WT, 440 bp), heterozygous (KI/WT, 550 and 440 bp), and homozygous (KI/KI, 550 bp) samples.

A model for integrin activation regulated by the membrane-proximal cytoplasmic salt bridge and generation of α4-R/AGFFKR mice in which the cytoplasmic salt bridge is disrupted. (A) A positively charged RGFFKR (a circled plus) forms the membrane-proximal salt bridge with a negatively charged residue(s) (a circled minus) in the β-subunit. The salt bridge facilitates clasping of the α/β cytoplasmic domains, maintaining the extracellular domains in a default inactive conformation (left). Disruption of the salt bridge, which is induced via binding of cytoplasmic proteins such as talin (not shown), triggers unclasping of the cytoplasmic domains, converting the extracellular domains to the active conformation (right). The α- and β-subunits are labeled in the extracellular domains. The plasma membrane is shown with 2 parallel dashed lines. (B) Amino acid sequence alignment of the membrane-proximal cytoplasmic domains of selected integrin α-subunits. A conserved GFFKR motif is highlighted. The arginine residues in the GFFKR motif (RGFFKR) are shown in bold. (C) Targeted insertion of the floxed ACN cassette containing both the neor gene and the Cre-recombinase gene under the control of the sperm-specific ACE promoter,25  mutated exon 28 (28**), and UTR into the Itga4 locus, followed by deletion of the ACN cassette. The configurations of the targeting vector, α4 integrin genomic locus, the targeted allele following homologous recombination, and the ACN-deleted allele are shown. Exons are displayed as black boxes and the floxed ACN cassette, UTR, and diphtheria toxin cassette (DT) are displayed as white boxes. loxP sites are displayed as triangles. The external probe containing exon 24, which was used to verify the targeting event, is indicated. The map displays the following restriction sites: E indicates EcoRI; S, ScaI; Sa, SacI; X, XbaI; and X*, engineered XbaI. (D) Genotyping and confirmation of the deleted ACN cassette by PCR. PCR bands are shown for wild-type (WT/WT, 440 bp), heterozygous (KI/WT, 550 and 440 bp), and homozygous (KI/KI, 550 bp) samples.

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