Membrane-associated guanylate kinase proteins MPP4 and MPP5 associate with Veli3 at distinct intercellular junctions of the neurosensory retina

Abstract

MPP4 and MPP5 are closely related members of the p55-subfamily of membrane-associated guanylate kinases (MAGUKs) known to mediate the assembly of protein complexes at the plasma membrane of cell-cell junctions. Both MPP4 and MPP5 have been implicated in retinal function; however, their specific roles in the cellular mechanisms underlying vision are largely unknown. Here, we generated specific poly-and monoclonal antibodies against the two proteins and show that MPP4 and MPP5 are localized at distinct sites of cell-cell contact in the mouse retina. While MPP4 is a component of the synaptic terminals of photoreceptors, MPP5 exclusively localizes to apical membrane domains of the outer limiting membrane (OLM) junctions. The vertebrate homologs of Caenorhabditis elegans lin-7, Veli1, -2, and -3, have previously been identified as putative binding partners of MPP5. In this study, we show that MPP4 directly interacts with the Veli proteins via L27 heterodimerization in vitro. In addition, two of the three Veli isoforms, Veli1 and -3, are demonstrated to be expressed in the mouse retina. Immunofluorescence microscopy reveals extensive colocalization of Veli3 with both MPP4 and MPP5. This association of Veli3 with either MPP4 or MPP5 suggests that the MAGUKs recruit Veli3 and its binding partners to different cellular regions of the retina where they may participate in the organization of specialized intercellular junctions.

TMEM16B, A Novel Protein with Calcium-Dependent Chloride Channel Activity, Associates with a Presynaptic Protein Complex in Photoreceptor Terminals

“…The full-length coding sequence of mouse Tmem16b was RT-PCR amplified from mouse retinal RNA with primer pair mA126-F10 (5Ј-GGGGTACCATCTGGGGACCAGCA CCATG-3Ј)/mA126-R13 (5Ј-CCGGTACCTCATACATTGGTGTGCT GGGACC-3Ј). PCR products were sequenced and cloned into the KpnI site of pcDNA3 (Invitrogen) or pCEP1.4 vector in-frame with a C-terminal rhodopsin (Rho)-1D4 tag (Stöhr et al, 2005) for expression in mammalian cells. The human and mouse cDNA sequences reported in this manuscript have been deposited in GenBank under the accession numbers FJ384095 and FJ384096.…”

“…A polyclonal anti-TMEM16B antiserum (TMEM16B-303) was generated in rabbits immunized with GST-TMEM16B-C-term fusion proteins (amino acids 950 -1002) and purified using HiTrap NHSactivated Sepharose HP columns (GE Healthcare) as described previously (Stöhr et al, 2005). For anti-TMEM16B monoclonal antibody production, mice were immunized with a GST-TMEM16B-N-term fusion protein (amino acids 14 -134) mixed with ImmunEasy Mouse Adjuvant (Qiagen).…”

“…Frozen eyecups were cryosectioned vertically at 10 m and fixed in 2% paraformaldehyde in 0.1 M PB for 10 min. Immunofluorescence labeling was performed as described previously (Stöhr et al, 2005). Each immunolabeling experiment was performed on eye sections from at least two animals.…”

“…Mammalian expression constructs of MPP4, VELI3, and PSD95 have been reported previously (Stöhr et al, 2005;Förster et al, 2009). Transfected 293-EBNA cells were solubilized in TBS (20 mM Tris pH 7.4, 0.15 M NaCl)/1% Triton X-100 supplemented with complete protease inhibitor cocktail tablets (Roche Applied Sciences) for 30 min and centrifuged at 16.000 ϫ g for 15 min at 4°C.…”

“…Recently, we and others have described a presynaptic protein complex in photoreceptors consisting of the cytoplasmic adaptor proteins MPP4, PSD95, and VELI3 that may also include PMCAs and the Ca 2ϩ -binding protein recoverin (Stöhr et al, 2005;Aartsen et al, 2006;Yang et al, 2007;Förster et al, 2009). MPP4 appears to hold a key scaffolding function in the recruitment and assembly of this complex as mice lacking MPP4 are characterized by an absence of PSD95, VELI3, and PMCAs from the ribbon synapse (Aartsen et al, 2006;Yang et al, 2007).…”