Expression and distribution of Trophoblast Glycoprotein in the mouse retina

Abstract

Abbreviations: CtBP2: C-terminal binding protein 2; GCL: ganglion cell layer; GPR179: G protein-coupled receptor 179; INL: inner nuclear layer; IPL: inner plexiform layer; LRR: leucinerich repeat; OPL: outer plexiform layer; PKCα: protein kinase C-alpha; RBC: rod bipolar cell; TPBG: trophoblast glycoprotein; TRPM1: transient receptor potential cation channel subfamily M member 1 Abstract We recently identified the leucine-rich repeat adhesion protein, trophoblast glycoprotein (TPBG), as a novel PKCa-dependent phosphoprotein in retinal rod bipolar cells (RBCs). Since TPBG has not been thoroughly examined in the retina, this study characterizes the localization and expression patterns of TPBG in the developing and adult mouse retina using two antibodies, one against the N-terminal, leucine-rich domain and the other against the C-terminal PDZinteracting motif. Both antibodies labeled dendrites and synaptic terminals of RBCs, as well as the cell bodies and dendrites of an uncharacterized class of amacrine cell. In transfected HEK293 cells, TPBG was localized to the plasma membrane and intracellular membranes and was associated with the tips of thin filopodia-like membrane projections. TPBG immunofluorescence in RBCs detected with the C-terminal antibody was strongly dependent on the activity state of the adult retina, with less labeling in dark-adapted compared to light-adapted retina, and less labeling in light-adapted PKCa knockout and TRPM1 knockout retinas compared to wild type, despite no change in total TPBG detected by immunoblotting. These results suggest that the C-terminal epitope is blocked in the dark-adapted and knockout retinas compared to light-adapted wild type retinas, possibly through interaction with a PDZ domain protein. During development, TPBG expression increases dramatically just prior to eye opening with a time course closely correlated with that of TRPM1 expression. In the retina, leucine-rich repeat proteins like TPBG have been implicated in the development and maintenance of functional bipolar cell synapses, and TPBG may play a similar role in RBCs.

Expression and distribution of trophoblast glycoprotein in the mouse retina

“…Mouse anti-CANA1S (1:4000; anti-GPR179; Abcam; Cat# ab2862; RRID: AB_2069567) is a monoclonal antibody raised against full-length native rabbit CACNA1S subunit purified from the rabbit muscle T-tubule dihydropyridine receptor. This CACNA1S antibody has been shown to strongly cross-react with retinal GPR179 in the tips of ON-bipolar cell dendrites (Hasan, Ray, & Gregg, 2016).…”

“…The copyright holder for this preprint (which was not this version posted March 27, 2019. ; https://doi.org/10.1101/590216 doi: bioRxiv preprint TPBG was first identified in trophoblasts (Hole & Stern, 1988) and has been mainly studied in embryonic development and in cancer (Barrow, Ward, Rutter, Ali, & Stern, 2005), where it is required for cell surface expression of CXCR4, a G-protein coupled receptor that mediates a chemotactic response to the CXCL12 chemokine (McGinn, Marinov, Sawan, & Stern, 2012;Southgate et al, 2010), and where it is diagnostic for metastasis and poor prognosis in cancer patients (Pukrop & Binder, 2008;Weeraratna et al, 2002). In mammalian embryonic cell lines, TPBG influences cytoskeletal organization and cell motility through modulation of Wnt signaling (Kagermeier-Schenk et al, 2011), and has also shown to bind to the PDZ domain of GIPC1, a scaffolding protein that regulates cell surface expression of G protein-coupled receptors (Awan et al, 2002).…”

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“…Indeed, TPBG silencing downregulated phosphorylated ERK and ACKR3. In the retina, TPBG is highly expressed by rod-BCs, where it has been shown to exhibit PRKCAdependent phosphorylation (Wakeham et al, 2020;Wakeham et al, 2019). TPBG phosphorylation was decreased in Prkca -/compared to wild-type mice.…”