The vitronectin receptor associates with clathrin-coated membrane domains via the cytoplasmic domain of its β5 subunit

Abstract

Rat myotubes cultured in fetal calf serum adhere to vitronectin-coated substrates through two distinct structures, focal contacts and clathrin-coated membrane domains. We studied the integrins in myotubes to learn how they associate with these two domains. Double label immunofluorescence studies with antibodies specific for clathrin, vinculin and several forms of integrin showed that focal contacts and clathrin-coated membrane domains contain both vitronectin receptors (VnR, containing beta-3 and beta-5integrins) and fibronectin receptors (FnR, containing beta1-integrin). VnR but not FnR associates tightly with the substrate in both domains, as the VnR alone remains attached to the coverslip when the lipid bilayer and other membrane proteins are removed by detergent. Ultrastructural studies confirmed the localization of the beta5 subunit of the VnR at both domains. We used intracellular injection and affinity chromatography to test the possibility that clathrin at coated membrane domains associates with the cytoplasmic sequence of the beta5 subunit of the VnR. Injection of a synthetic peptide containing the NPXY motif from the cytoplasmic domain of the human beta5 subunit, SRARYEMASNPLYRKPIST, depleted clathrin from coated membrane domains without affecting clathrin in perinuclear structures or vinculin at focal contacts. Injection of the homologous beta1 peptide, MNAKWDTGENPIYKSAVITT, also containing an NPXY motif, had no significant effect on any of these structures. Affinity matrices containing the beta5 but not the beta1 peptide selectively retained clathrin from myotube extract, and bound clathrin could be selectively eluted by soluble forms of the beta5 but not the beta1 peptide. Thus, a sequence including the NPXY motif in the integrin beta5 subunit is involved in the specific anchoring of the VnR, but not the FnR, to clathrin-coated membrane.

The structure and spontaneous curvature of clathrin lattices at the plasma membrane

“…Cilengitide selectively inhibits aVb5-integrin and aVb3-integrin connections with the extracellular matrix (Reardon and Cheresh, 2011). aVb5integrin has been shown to localize to flat clathrin structures (De Deyne et al, 1998;Zuidema et al, 2018). We show that removal of this connection vastly decreases the amount of flat clathrin in HeLa cells.…”

“…Because clathrin plaques are less prevalent on unadhered surfaces (Saffarian et al, 2009;Grove et al, 2014), we reasoned that physical adhesion to the substrate could impart a flattening force. b5integrins have been shown to localize to clathrin plaques (De Deyne et al, 1998;Zuidema et al, 2018). Figures 4D and 4G-4I show that disrupting the connection between b5-integrins and the extracellular matrix with the drug cilengitide (CTA) caused a 2-fold loss of flat structures in HeLa cells (0.96% ± 0.35% with CTA versus 2.1% ± 1.8% membrane occu-pation in control).…”

“…Adjacent to its role in endocytosis, flat clathrin lattices have also been proposed to play other important physiological roles. For example, flat clathrin can act as an integrin-containing adhesion site (De Deyne et al, 1998), connecting retraction fibers to the extracellular substrate during mitosis, (Lock et al, 2018) adhering to engulfed particles during phagocytosis (Aggeler and Werb, 1982), gripping contractile fibers in skeletal muscle (Vassilopoulos et al, 2014;Franck et al, 2019), and aiding cell motility (Zuidema et al, 2018;Elkhatib et al, 2017;Baschieri et al, 2018). Furthermore, an increase of flat clathrin during signaling has suggested that it could act as a dynamic signaling platform (Leyton-Puig et al, 2017).…”

The crosstalk between microtubules, actin and membranes shapes cell division

“…Two distinct types of clathrin-coated structures (CCSs) can be visualized at the PM of mammalian cells: the dynamic curved CCPs and the large, long-lived, flat clathrin lattices, called 'coated plaques' (figure 3), first observed several decades ago [85][86][87][88][89][90]. The latter structures are very stable, enriched in signalling receptors (e.g.…”

Improved Boron Neutron Capture Therapy Using Integrin αvβ3-Targeted Long-Retention-Type Boron Carrier in a F98 Rat Glioma Model

“…Although the interaction between RGD and integrins has long been known, the mechanism of internalization by binding of RGD peptides to integrins has not yet been elucidated. However, it has been reported that at least the endocytosis of integrin α v β 3 is mediated via clathrin-dependent endocytosis or uncoated vesicles [ 49 , 50 ]. Schraa et al showed that internalization of monomeric RGD ligands is independent of their α v β 3 receptor and occurs via a liquid-phase endocytic pathway.…”