Extraribosomal functions associated with the C terminus of the 37/67 kDa laminin receptor are required for maintaining cell viability

J Scheiman,D Meruelo,J Ziello,J-C Tseng,K V Jamieson

doi:10.1038/cddis.2010.19

Abstract

The 37/67 kDa laminin receptor (LAMR) is a multifunctional protein, acting as an extracellular receptor, localizing to the nucleus, and playing roles in rRNA processing and ribosome assembly. LAMR is important for cell viability; however, it is unclear which of its functions are essential. We developed a silent mutant LAMR construct, resistant to siRNA, to rescue the phenotypic effects of knocking down endogenous LAMR, which include inhibition of protein synthesis, cell cycle arrest, and apoptosis. In addition, we generated a C-terminal-truncated silent mutant LAMR construct structurally homologous to the Archaeoglobus fulgidus S2 ribosomal protein and missing the C-terminal 75 residues of LAMR, which displays more sequence divergence. We found that HT1080 cells stably expressing either silent mutant LAMR construct still undergo arrest in the G1 phase of the cell cycle when treated with siRNA. However, the expression of full-length silent mutant LAMR rescues cell viability, whereas the expression of the C-terminal-truncated LAMR does not. Interestingly, we also found that both silent mutant constructs restore protein translation and localize to the nucleus. Our findings indicate that the ability of LAMR to regulate viability is associated with its C-terminal 75 residues. Furthermore, this function is distinct from its role in cell proliferation, independent of its ribosomal functions, and may be regulated by a nonnuclear localization.

Highlights

The 37/67 kDa laminin receptor (LAMR), as a cell surface receptor, has roles in cell migration,[1] invasion,[2] angiogenesis,[3] and extracellular matrix remodeling.[4,5] LAMR serves as a cellular receptor for many pathogens, including prion protein,[6] bacteria,[7] and numerous viruses.[8,9,10,11] Since its initial discovery, LAMR was determined to be a highly conserved ribosomal protein that acquired its extracellular functions during evolution.[12]
We performed experiments with all selected cell lines, yielding similar results, we report our findings with one of each. silent mutant LAMR (silMUT), silMUT220, and parental HT1080 (WT) cells were transfected with either siLAMR or a fluorescently labeled nontargeting control siRNA and analyzed for total LAMR protein expression
Exogenous silMUT LAMR, detected by both an antibody for LAMR (Figure 2a, arrow) and an antibody for the FLAG tag (Figure 2b, middle), is not reduced by siLAMR treatment but rather appears to be increased in expression compared with siGLO-treated silMUT cells. silMUT220 is not readily detected by the antibody we used for LAMR as it is missing a part of its C-terminal epitope

Summary

Introduction

The 37/67 kDa laminin receptor (LAMR), as a cell surface receptor, has roles in cell migration,[1] invasion,[2] angiogenesis,[3] and extracellular matrix remodeling.[4,5] LAMR serves as a cellular receptor for many pathogens, including prion protein,[6] bacteria,[7] and numerous viruses.[8,9,10,11] Since its initial discovery, LAMR was determined to be a highly conserved ribosomal protein that acquired its extracellular functions during evolution.[12]. It may have enabled LAMR to become a Received 12.2.10; revised 24.3.10; accepted 31.3.10; Edited by RA Knight dimeric molecule, as the 37 kDa LAMR has been shown to serve as a precursor to the 67 kDa LAMR,[25] which has only been observed in vertebrates and is dependent on posttranslational modifications.[26,27] Extraribosomal functions associated with the C terminus of LAMR may have enabled alternative mechanisms to regulate cell viability in higher organisms, including nuclear localization. We found protein translation to be dramatically reduced as knockdown of LAMR in HT1080 cells leads to fewer 40 s subunits and 80 s monosomes, as well as an increase in unassociated 60 s subunits. HT1080 cells with reduced LAMR expression have an impaired ability to grow tumors in vivo

Methods

Results

Conclusion