Study of multiple roles of the 37/67 kDa laminin receptor in cellular function

Abstract

The 37/67 kDa laminin receptor (LAMR) is an extracellular receptor, localizes to the nucleus, and plays roles in rRNA processing and ribosome assembly. LAMR is ubiquitously expressed and highly conserved as a member of the S2 ribosomal family, having acquired extracelluar functions through sequence divergence. LAMR is important for cell viability, however it is unclear which of its functions are essential. Additionally, LAMR is overexpressed in many types of cancer, playing roles in tumor cell migration and invasion. Studying LAMR is therefore important from both a cellular functioning and therapeutic standpoint. We found that LAMR is vital for tumor cell proliferation, survival, and protein translation. siRNA mediated reduction in expression of LAMR in HT1080 human fibrosarcoma cells leads to G1 phase cell cycle arrest by altering Cyclin A2/B1, CDK1/2, Survivin, and p21 expression levels. HT1080 cells treated with siRNA become morphologically distinct and lose mitochondrial membrane potential, developing a sub-G1/apoptoic population. Further, reduction in LAMR expression leads to a dramatic decrease in newly synthesized proteins due to reduction in 40S subunits and 80S monosomes, and an increase in unassociated 60S ribosomal subunits. Ultimately, reduction in LAMR expression inhibits HT1080 tumor development in vivo. To clarify which functions of LAMR are responsible for maintaining viability, we developed a silent mutant LAMR construct resistant to siRNA to rescue the phenotypic effects of knocking down endogenous LAMR. Additionally, we generated a C-terminal truncated silent mutant LAMR construct structurally homologous to the A. fulgidus S2 ribosomal protein and missing the C-terminal 75 amino acids of LAMR, which displays more sequence divergence. We found 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, expression of full-length silent mutant LAMR rescues cell viability while 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. Further, this function is distinct from its role in cell proliferation, independent of its ribosomal functions, and may be regulated by a non-nuclear localization