Cupidin, an isoform of Homer/Vesl, interacts with the actin cytoskeleton and activated rho family small GTPases and is expressed in developing mouse cerebellar granule cells

Abstract

A developmentally regulated Homer/Vesl isoform, Cupidin (Homer 2a/Vesl-2Delta11), was isolated from postnatal mouse cerebellum using a fluorescent differential display strategy. The strongest expression of Cupidin was detected in the cerebellar granule cells at approximately postnatal day 7. Cupidin was enriched in the postsynaptic density fraction, and its immunoreactivity was concentrated at glomeruli of the inner granular layer when active synaptogenesis occurred. Cupidin protein could be divided into two functional domains: the N-terminal portion, which was highly conserved among Homer/Vesl family proteins, and the C-terminal portion, which consisted of a putative coiled-coil structure, including several leucine zipper motifs. The N-terminal fragment of Cupidin, which was able to associate with metabotropic glutamate receptor 1 (mGluR1), also interacted with F-actin in vitro. In keeping with this, F-actin immunocytochemically colocalized with Cupidin in cultured cerebellar granule cells, and a Cupidin-mGluR1-actin complex was immunoprecipitated from crude cerebellar lysates using an anti-Cupidin antibody. On the other hand, the C-terminal portion of Cupidin bound to Cdc42, a member of Rho family small GTPases, in a GTP-dependent manner in vitro, and Cupidin functionally interacted with activated-Cdc42 in a heterologous expression system. Together, our findings indicate that Cupidin may serve as a postsynaptic scaffold protein that links mGluR signaling with actin cytoskeleton and Rho family proteins, perhaps during the dynamic phase of morphological changes that occur during synapse formation in cerebellar granule cells.

Fig. 1.

Isolation, detection, and expression pattern of clone 13–2/Cupidin during cerebellar development and in various tissues. A, FDD analysis. Cupidin was first detected as a developmentally induced band named clone 13–2, indicated by anarrowhead. B, RT-PCR analysis in the developing cerebellum. RT-PCR for Cupidin was performed using an inner primer set corresponding to the internal sequences of the cloned FDD band. GAPDH was used as an RT-PCR control. C, Poly(A)⁺ RNA (10 μg) was transferred onto a nylon membrane. ³²P-labeled full-length Cupidin cDNA was used as a probe. As a control, a ³²P-labeled GAPDH cDNA was used.D, RT-PCR analysis in various tissues of P7 mice.E, In situ localization of clone 13–2/Cupidin mRNA in the mouse cerebellum. Parasagittal sections of cerebella at P7 and adult stage were probed with the digoxigenin-labeled antisense cRNA probe prepared using a fragment (647–1220 nt) of Cupidin cDNA as a template. The antisense probe recognized two splicing variant forms of Cupidin. Specificity of labeling signals was confirmed using the sense probe (data not shown).PL, Purkinje cell layer. Magnification, 400×. Scale bar, 100 μm.

Fig. 2.

Schematic structure of Cupidin and the specificity of anti-Cupidin antibody. A, Schematic representation of the structure of Cupidin and its truncation mutants. CPD is characterized by two major regions: an EVH1-like region, including a single PDZ consensus motif, and a Citron-like coiled-coil region, including leucine zippers. CPD/N (aa 1–111) and CPD/C (aa 112–343) were constructed by deleting the C-terminal portion and the N-terminal portion, respectively. B, Sequence alignment of Cupidin with Citron, a putative target protein for Rho GTPase. Identical amino acids are shaded. The Rho-binding region of Citron isunderlined. The C-terminal region of Cupidin (Leu-112 to Leu-336) exhibited a weak identity (22%) with a portion of Citron, including its Rho-binding region. C, Affinity-purified anti-Cupidin antibody specifically recognized a doublet band (44 and 46 kDa) in lysates of P7 mouse cerebellum (20 μg). Thebottom 44 kDa band comigrated with Cupidin (CPD-α) expressed in HeLa cells. The top 46 kDa band comigrated with a splice variant of Cupidin (CPD-β) expressed in HeLa cells. The antibody did not recognized other Homer family members, Homer 1c and Homer 3.

Fig. 3.

Cupidin interacts with Rho family small GTPases. A, Direct binding of RhoA, Rac1, and Cdc42 to GST-fused Cupidin in a ligand overlay assay. GST-CPD (lane 1), GST-CPD/N (lane 2), GST-CPD/C (lane 3), GST-Citron (lane 4), and GST (lane 5) were separated by SDS-PAGE, blotted onto nitrocellulose membranes, and probed with [³⁵S]GTPγS-bound (top) or [³⁵S]GDPβS-bound (bottom) forms of each GST-fused Rho family GTPases (RhoA, RhoA^A37, Rac1, Cdc42), as indicated at thetop. B, In vitro binding of Cupidin to Cdc42 in a coimmunoprecipitation assay. GST, GST-RhoA, GST-Rac1, and GST-Cdc42, loaded with either GTPγS or GDP, were mixed with P7 mouse cerebellar lysates. Each aliquot was immunoprecipitated with either the anti CPD antibody (CPD Ab) or preimmune serum (pre) and was analyzed by Western blot using the anti-GST antibody. Loaded nucleotides and composition of the analyzed mixtures are indicated at the top of each lane.Input, The equivalent mixture without being subjected to immunoprecipitation. C, A cross-link assay of Cupidin. GST, GST-RhoA, GST-Rac1, and GST-Cdc42, loaded with either GTPγS or GDP, were mixed with GST-free Cupidin protein. Each aliquot was treated with the cross-linker DMP and analyzed by Western blotting using the anti-GST antibody. Loaded nucleotides and the compo-sition of the analyzed mixture are indicated at the top of eachlane. Note that, by cross-linking in the presence of CPD, a part of Cdc42 loaded with GTPγS (GTPγS-Cdc42) enters a higher molecular weight complex, indicated byarrows.

Fig. 4.

Cupidin overexpression affects Cdc42^V12 phenotype in HeLa cells. Overexpression of CPD alone (left panels), myc tagged-Cdc42^V12 alone (middle panels), or both (right panels) in HeLa cells. Transfected cells were stained using the anti CPD antibody (FITC), anti-myc antibody (Cy5), and Texas Red-phalloidin. Top panels are superimposed composite images using three pseudocolors (green for CPD, redfor phalloidin, and blue for myc tagged-Cdc42^V12). Note that filopodia-like microspike structures were visible in Cdc42^V12-overexpressed cells, as indicated byarrows. However, few such structures were induced in cells overexpressing either CPD alone or CPD and Cdc42^V12. Scale bar, 60 μm.

Fig. 5.

Cupidin interacts with F-actin and mGluR1α. A, F-actin binding to Cupidin in a cosedimentation assay. GST, GST-CPD/N, and GST-CPD/C were incubated with (+) or without (−) F-actin prepared from chicken skeletal muscles and then centrifuged. Equivalent protein amounts of the supernatant (S) and pellet (P) fractions were separated by SDS-PAGE and stained with Coomassie Brilliant Blue (CBB). A representative result of five independent experiments is shown here. The arrowsindicate the position of actin, and the arrowheadsrepresent each GST-fusion protein. B, Binding of Cupidin to mGluR1α in a pull down assay. The S1 fraction prepared from P7 mouse cerebellum or cerebrum was incubated with GST-CPD/N protein and then immobilized onto glutathione-Sepharose beads. After extensive washing, GST-CPD/N-bound proteins were extracted with SDS-PAGE sample buffer and were analyzed by Western blotting using an anti-mGluR1α polyclonal antibody. Lane 1, Input (the same amounts of lysates used for the assay were loaded); lane 2, eluate from a GST-bound column; lane 3, eluate from a GST-CPD/N bound column. C, Coimmunoprecipitation of both mGluR1 and actin from P7 mouse cerebellar lysates using the anti-CPD antibody. The immunoprecipitates obtained with either the preimmune serum (lane 2) or the affinity-purified anti-CPD polyclonal antibody were examined by Western blotting with the indicated antibodies (anti-CPD, anti-actin, and anti-mGluR1α). Lane 1 is the detergent extract of P7 mouse cerebellum. Thearrow indicates Cupidin signal, and theasterisk indicates the heavy chain of IgG.D, Primary-cultured mouse cerebellar granule neurons at 7 DIV were triple-stained with an anti-CPD polyclonal antibody (FITC;a), Texas Red-phalloidin (b), and an anti-synaptophysin monoclonal antibody (Cy5; c) and observed by confocal microscopy.d is a superimposed composite images ofa–c using three pseudocolors (green for CPD, red for phalloidin, and blue for synaptophysin).Arrows indicate representative positions at which the three pseudocolors overlapped. Scale bar, 10 μm.

Fig. 6.

Localization of Cupidin overexpressed in MDCK cells at sites of cell–cell contact. MDCK cells overexpressing GFP-CPD (left panels), GFP-CPD/C (middle panels), and GFP-CPD/N (right panels) were stained with Texas Red-phalloidin. Middle panels show GFP images,bottom panels show F-actin images overlapping cell–cell contact sites, and top panels show the merged images. Note that overexpressed GFP-CPD specifically concentrated at cell–cell contact sites, as indicated by arrows, whereas both overexpressed GFP-CPD/N and GFP-CPD/C widely localized throughout the cytoplasm. Scale bars, 10 μm.

Fig. 7.

The cellular and subcellular distribution of Cupidin. (A, A′, B,B′) A sagittal cerebellar section of P7 mouse was immunostained with an anti-Cupidin antibody (A,A′) and an anti-synaptophysin antibody (B, B′). Magnification: A, B, 400×; A′, B′, 1000× of IGL region. Examples that Cupidin signals could be overlapped with synaptophysin signals are indicated by arrows.PL, Purkinje cell layer. C, Crude synaptosomes from P7 mouse cerebellum were prepared by differential centrifugation (P2′), lysed hypotonically, and fractionated into heavy membranes (LP1), synaptic vesicles (LP2), and cytosol (LS2). PSD fractions were prepared from LP1 fractions. Proteins (25 μg) from each fraction were subjected to Western blot using the antibodies against the indicated proteins. An arrow indicates the signal of Cupidin.

Fig. 8.

Synaptic expression of Cupidin in granule cells during the development of the cerebellar cortex. A, Western blot analysis for developmental expression of Cupidin in the cerebellum. Whole lysates of mouse cerebellum at various stages were examined by Western blotting using the anti-CPD antibody. The expression of the top 46 kDa signal persisted even after P7, whereas the expression of the bottom 44 kDa Cupidin was peaked at approximately P7 and then was hardly detectable in adult lysates. B, Sagittal sections of P7, P14, and adult mouse cerebella were immunostained with the anti-CPD antibody (α-CPD Ab.; top panels) and anti-synaptophysin antibody (α-Syn. Ab.; bottom panels), followed by HRP-based DAB–H₂O₂ reaction. Apparently, a high intensity of CPD signals in the IGL was prominent at P7 and then diminished onwards. In contrast, synaptophysin signals in IGL were increased both in size and intensity, indicating the maturation of glomeruli. PL, Purkinje cell layer. Magnification, 400×. Scale bar, 40 μm. C, Primary cultured mouse cerebellar granule neurons at 7, 14, and 21 DIV were costained with the anti-CPD antibody (α-CPD Ab.; left panels) and the anti-synaptophysin antibody (α-Syn. Ab.; right panels) and observed using confocal microscopy. Note that a large number of punctate CPD-signals again peaked at 7 DIV, in contrast to the lasting increase in synaptophysin-signals until 21 DIV. Scale bar, 50 μm.