Deficits in receptor-mediated endocytosis and recycling in cells from mice bearing a disruption of the Gpr107 locus

Abstract

GPR107 is a type III integral membrane protein that was initially predicted to be a member of the family of G-protein-coupled receptors. This report shows that deletion of Gpr107 leads to an embryonic lethal phenotype that is characterized by a reduction in cubilin transcript abundance and a decrease in the representation of multiple genes implicated in the cubilin-megalin endocytic receptor complex (megalin is also known as LRP2). Gpr107-null fibroblast cells exhibit reduced transferrin internalization, decreased uptake of low-density lipoprotein (LDL) receptor-related protein-1 (LRP1) cargo and resistance to toxins. Colocalization studies and proteomic analyses suggest that GPR107 associates with clathrin and the retromer protein VPS35 and that GPR107 might be responsible for the return of receptors to the plasma membrane from endocytic compartments. The highly selective deficits observed in Gpr107-null cells indicate that GPR107 interacts directly or indirectly with a limited subset of surface receptors.

Post-Golgi anterograde transport requires GARP-dependent endosome-to-TGN retrograde transport

“…Overexpression of TMEM87B, a close homologue of TMEM87A and a member of the same family, also restored the defect in V54KO cells ( Figure 6A ; geometric means of cell surface levels of VFG-GPI and FVG-TM were 1.85 ± 0.22 and 3.42 ± 0.99 times, respectively, those in V54KO+Vec; n = 3), suggesting its functional redundancy with TMEM87A. Recently a role for GPR107—another LU7TM family member—was shown in retrograde transport of toxins such as exotoxin A of Pseudomonas aeruginosa and ricin ( Carette et al ., 2011a ; Elling et al ., 2011 ; Tafesse et al ., 2014 ; Zhou et al ., 2014 ). Unlike TMEM87A and TMEM87B, overexpression of GPR107 or GPR108, a member of LU7TM family closely related to GPR107, did not restore the delayed transport of either VFG-GPI or FVG-TM in V54KO cells ( Figure 6B ; geometric means of cell surface levels of VFG-GPI and FVG-TM in V54KO+GPR107 were 1.21 ± 0.38 and 0.98 ± 0.11 times, respectively, those in V54KO+Vec [ n = 2], and those in V54KO+GPR108 were 1.23 ± 0.14 and 1.15 ± 0.027 times, respectively, those in V54KO+Vec [ n = 2]).…”

GPR108, an NF-κB activator suppressed by TIRAP, negatively regulates TLR-triggered immune responses

“…Although Gpr108 bears structural similarity to G-protein coupled receptors, to date no study has demonstrated a functional role for the protein. The related gene Gpr107 participates in the regulation of receptor-mediated endocytosis [ 10 ]. In this study Gpr108 -null mice did not show any gross phenotype whereas Gpr107 -null mice exhibited embryonic lethality.…”

“…The targeting BAC strategy used to generate Gpr108 -null mice has been previously described [ 10 , 11 ]. The targeted clones MT1 and MT2 were screened by MLPA using multiplex MLPA probes ( S1 Table ).…”

Mouse embryonic fibroblasts exhibit extensive developmental and phenotypic diversity

“…One practice that can protect against this is the use of two sublines of a cloned fibroblast line from a null mouse, one subline consisting of the original (null) clone of fibroblast cells transduced with an empty retroviral vector and the other consisting of the null cell clone transduced with a retroviral vector that restores the missing gene product (44). If precautions of this type are not taken, there is a possibility that the variation observed between embryonic fibroblasts isolated from different mice could reflect the properties of different populations of fibroblasts, imperiling the conclusions reached.…”