Pg. Dedeyne et al., THE VITRONECTIN RECEPTOR ASSOCIATES WITH CLATHRIN-COATED MEMBRANE DOMAINS VIA THE CYTOPLASMIC DOMAIN OF ITS BETA(5) SUBUNIT, Journal of Cell Science, 111, 1998, pp. 2729-2740
Rat myotubes cultured in fetal calf serum adhere to vitronectin-coated
substrates through two distinct structures, focal contacts and clathr
in-coated membrane domains. We studied the integrins in myotubes to le
arn how they associate with these two domains. Double label immunofluo
rescence studies with antibodies specific for clathrin, vinculin and s
everal forms of integrin showed that focal contacts and clathrin-coate
d membrane domains contain both vitronectin receptors (VnR, containing
beta(3)- and beta(5)-integrins) and fibronectin receptors (FnR, conta
ining beta(1)-integrin), VnR but not FnR associates tightly with the s
ubstrate in both domains, as the VnR alone remains attached to the cov
erslip when the lipid bilayer and other membrane proteins are removed
by detergent, Ultrastructural studies confirmed the localization of th
e beta(5) subunit of the VnR at both domains. We used intracellular in
jection and affinity chromatography to test the possibility that clath
rin at coated membrane domains associates with the cytoplasmic sequenc
e of the beta(5) subunit of the VnR, Injection of a synthetic peptide
containing the NPXY motif from the cytoplasmic domain of the human bet
a(5) subunit, SRARYEMASNPLYRKPIST, depleted clathrin from coated membr
ane domains without affecting clathrin in perinuclear structures or vi
nculin at focal contacts. Injection of the homologous beta(1) peptide,
MNAKWDTGENPIYKSAVITT, also containing an NPXY motif, had no significa
nt effect on any of these structures. Affinity matrices containing the
beta(5) but not the beta(1) peptide selectively retained clathrin fro
m myotube extract, and bound clathrin could be selectively eluted by s
oluble forms of the beta(5) but not the beta(1) peptide. Thus, a seque
nce including the NPXY moth in the integrin beta(5) subunit is involve
d in the specific anchoring of the VnR, but not the FnR, to clathrin-c
oated membrane.