A tyrosine-based sorting signal in the beta2 integrin cytoplasmic domain mediates its recycling to the plasma membrane and is required for ligand-supported migration.
Integrins play pivotal roles in supporting shear- and mechanical-stress-resistant cell adhesion and migration. These functions require the integrity of the short beta subunit cytoplasmic domains, which contain multiple, highly conserved tyrosine-based endocytic signals, typically found in receptors undergoing regulated, clathrin-dependent endocytosis. We hypothesized that these sequences may control surface integrin dynamics in statically adherent and/or locomoting cells via regulated internalization and polarized recycling of the receptors. By using site-directed mutagenesis and ectopic expression of the alphaL/beta2 integrin in Chinese hamster ovary cells, we found that Y735 in the membrane-proximal YRRF sequence is selectively required for recycling of spontaneously internalized receptors to the cell surface and to growth factor-induced membrane ruffles. Disruption of this motif by non-conservative substitutions has no effect on the receptor's adhesive function, but diverts internalized integrins from a recycling compartment into a degradative pathway. Conversely, the non-conservative F754A substitution in the membrane-proximal NPLF sequence abrogates ligand-dependent adhesion and spreading without affecting receptor recycling. Both of these mutants display a severe impairment in ligand-supported migration, suggesting the existence in integrin cytoplasmic domains of independent signals regulating apparently unrelated functions that are required to sustain cell migration over specific ligands.
Fabbri
M
M
Scientific Institute San Raffaele-DIBIT, University of Milano School of Medicine, via Olgettina 58, I-20132 Milano, Italy.
Fumagalli
L
L
Bossi
G
G
Bianchi
E
E
Bender
J R
JR
Pardi
R
R
D000595:Amino Acid Sequence; D000818:Animals; D018821:CD18 Antigens / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D002448:Cell Adhesion; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002465:Cell Movement; D006224:Cricetinae; D003593:Cytoplasm / Q000737:chemistry; D004705:Endocytosis; D004815:Epidermal Growth Factor / Q000494:pharmacology; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014443:Tyrosine / Q000737:chemistry
D018821:CD18 Antigens; D008024:Ligands; D011994:Recombinant Proteins; D014443:Tyrosine; D004815:Epidermal Growth Factor