Formation of the 67-kDa laminin receptor by acylation of the precursor

Abstract

Even though the involvement of the 67-kDa laminin receptor (67LR) in tumor invasiveness has been clearly demonstrated, its molecular structure remains an open problem, since only a full-length gene encoding a 37-kDa precursor protein (37LRP) has been isolated so far. A pool of recently obtained monoclonal antibodies directed against the recombinant 37LRP molecule was used to investigate the processing that leads to the formation of the 67-kDa molecule. In soluble extracts of A431 human carcinoma cells, these reagents recognize the precursor molecule as well as the mature 67LR and a 120-kDa molecule. The recovery of these proteins was found to be strikingly dependent upon the cell solubilization conditions: the 67LR is soluble in NP-40-lysis buffer whereas the 37LRP is NP-40-insoluble. Inhibition of 67LR formation by cerulenin indicates that acylation is involved in the processing of the receptor. It is likely a palmitoylation process, as indicated by sensitivity of NP-40-soluble extracts to hydroxylamine treatment. Immunoblotting assays performed with a polyclonal serum directed against galectin3 showed that both the 67- and the 120-kDa proteins carry galectin3 epitopes whereas the 37LRP does not. These data suggest that the 67LR is a heterodimer stabilized by strong intramolecular hydrophobic interactions, carried by fatty acids bound to the 37LRP and to a galectin3 cross-reacting molecule.

Different Isoforms of the Non-Integrin Laminin Receptor Are Present in Mouse Brain and Bind PrP

“…Our data strongly support a physiological role of the laminin receptor/The polypeptide predicted from the non-integrin laminin receptor cDNA sequence consists of 295 amino acids and the in vitro translation of selectively hybridized mRNA produced a protein with an apparent molecular mass of 37 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Rao et al, 1989). However, other isoforms of the laminin receptor, displaying higher molecular masses, have also been described (Castronovo et al, 1991;Landowski et al, 1995;Menard et al, 1997;Buto et al, 1998). One of these isoforms, the 67 kDa high affinity laminin receptor (LR: presumably the mature form of the receptor), is thought to arise from the heterodimerization of its precursor molecule, the 37 kDa LRP, with a still unidentified molecule (Buto et al, 1998).…”

“…However, other isoforms of the laminin receptor, displaying higher molecular masses, have also been described (Castronovo et al, 1991;Landowski et al, 1995;Menard et al, 1997;Buto et al, 1998). One of these isoforms, the 67 kDa high affinity laminin receptor (LR: presumably the mature form of the receptor), is thought to arise from the heterodimerization of its precursor molecule, the 37 kDa LRP, with a still unidentified molecule (Buto et al, 1998). The 67 kDa LR is believed to be the functional isoform of the receptor regarding its ability to mediate strong attachement of cells to laminin (Lesot et al, 1983;Malinoff and Wicha, 1983;Rao et al, 1983), to be overexpressed on cancer cell sufaces, and to promote the invasive and metastatic capacity of these cells (Menard et al, 1997).…”

The matrix metalloproteinase stromelysin-3 cleaves laminin receptor at two distinct sites between the transmembrane domain and laminin binding sequence within the extracellular domain

“…Although LR has no obvious signal peptide and precursor of LR remained in cytoplasm as a component of the translational machinery [39], cell surface association of mature LR has been well documented [40,41]. It is unclear how the 37 kD precursor is converted to the 67 kD LR, although it has been suggested that it is due to either homo or heterodimerization of the 37 kD LR [21,22]. Regardless, in the membrane bound state, the C-terminal two-thirds of the LR is located extracellularly and contains a 6 amino acid laminin-binding sequence, whereas the N-terminal third faces the cytoplasm preceded by a short transmembrane domain [40].…”

“…The two cleavage sites by ST3 are indicated by two arrows. LR is either a homodimer of the 37 kD or a heterodimer with another protein [21,22]. Only the 37 kD precursor protein (referred here and in the text simply as LR) is shown here for simplicity.…”

“…Thus, to understand the function of ST3 in vivo, it is critical to identify the physiological substrates of ST3. Toward this end, we employed the yeast two-hybrid screen to isolate proteins that interact with ST3 by using mRNAs from metamorphosing intestine and identified the 37 kD precursor of the 67 kD laminin receptor as a potential physiological substrate of Xenopus ST3 (the 67 kD laminin receptor is reported to consist of a homodimer of the 37 kD precursor or a heterodimer of this precursor with an, as yet, unknown partner [21,22]. For simplicity, we will refer to both as LR in this paper).…”