The developmental regulation of CD81 in the rat retina

Abstract

Purpose: The tetraspanin CD81 is expressed in Müller glial cells and retinal pigment epithelium (RPE). CD81 and other members of the tetraspanin family link extracellular interactions of cells into intracellular cascades. This study examined the developmental expression of CD81 and protein-protein interactions linking CD81 to intracellular proteins.

Methods: We used synthetic peptides of the C-terminal intracellular domains of CD81 to probe fusion proteins of PDZ domains blotted to nitrocellulose membranes, then confirmed the relationships between the PDZ proteins using immunoprecipitation methods. Colocalization of the associated proteins was analyzed across development, using double-label immunohistochemical methods in the retina of Sprague-Dawley rats.

Results: The C-terminal intracellular sequences of CD81 bound to three putative PDZ domains that potentially represented domains on Sap97 and EBP50. In immunoprecipitation experiments using RPE cells, CD81 coprecipitated with both proteins, EBP50 and Sap97. Like CD81, EBP50 and Sap97 are expressed at low levels immediately after birth and upregulated during the first two postnatal weeks, reaching almost adult levels at postnatal day 20. In the RPE layer, synapse-associated protein 97 (Sap97) and CD81 were associated with the basolateral surface of the cells; ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) localizing with CD81 was found on microvilli at the inner surface of RPE cells.

Conclusions: These results support the hypothesis that CD81 is associated with the final stages of RPE cell maturation, establishing key molecular interactions linking the cell membrane proteins into macromolecular complexes containing PDZ protein scaffolds.

Figure 1

Binding of C-terminal peptides to PDZ domains. The binding of synthetic peptides to dot-blotted GST fusion proteins representing PDZ domains. Lane 1: a peptide that models the C-terminus of CD81 Lane 2: a peptide without the putative PDZ binding domain. Lane 3: the binding of a scrambled protein. Specific binding was observed for the Sap97 domain 3, the protein-tyrosine phosphatase domain 1 (PTP-D1), and the X11L2 protein domain 1 (Mint-D1). Each PDZ domain is spotted with two concentrations of protein: 400 ng (upper spot) and 80 ng (lower spot). GST represents glutathione S-transferase.

Figure 2

Proteins associated with CD81. Cultured rat retinal pigment epithelium (RPE) cells were solubilized in 1% Brij 97 and immunoprecipitated with mouse anti-rat CD81 antibody. The sample of proteins was then separated by SDS PAGE, transferred to nitrocellulose, and probed with antibodies directed against CD81 (lane A), EBP50 (lane B), Ezrin (lane C), and Sap97 (lane D). Note that CD81 coimmunoprecipitated with EBP50, Ezrin, and Sap97. In control experiments, antibodies directed against the transferrin receptor (CD71) were used to immunoprecipitate proteins from cultured RPE. When these samples were probed for EBP50 (lane E) or Sap97 (lane F), these two PDZ proteins were not observed. Molecular weight markers are indicated to the left in kDa.

Figure 3

Quantitative changes in CD81 and EBP50. The developmental regulation of EBP50 and CD81 is shown for different developmental ages: postnatal day 0 (P0), P2, P10, P15, P20, and adult. At P0, scant EBP50 and CD81 are present in the retina. There is a gradual increase until P20, when almost adult levels are observed. The levels of these two proteins do not completely mirror each other. EBP50 is expressed only in the microvilli of the retinal pigment epithelium cells, and CD81 is expressed throughout the entire retina.

Figure 4

Colocalization of CD81 with Sap97 and EBP50 in the developing retinal pigment epithelium layer. The developmental pattern of expression of Sap97 (A, D, G, J), EBP50 (M, P, S, V), and CD81 (B, E, H, K, N, Q, T, W) is shown for the retinal pigment epithelium (RPE) cell layer in double-stained sections at different developmental ages: P0 (A-C, M-O), P2 (D-F, P-R), P10 (G-I, S-U), and P15 (J-L, V-X). The merged images are shown to the right (C, F, I, L, O, R, U, X). At P0 (A-C, M-O) CD81 levels are low, with only a faint outline of the developing RPE. There is little immunostaining for Sap97 (A) or EBP50 (M). By P2 (D-F, P-R), CD81 defined the cellular membranes of the RPE cells. There is a clear upregulation of Sap97 along the basal surface of the RPE (D, arrow). Sap97 is distributed on the basolateral surfaces of the RPE cells (F). The levels of EBP50 are also increasing (P), forming a tight band of immunoreactivity at the junction between the RPE cells and the developing photoreceptors (arrow). EBP50 immunoreactivity labels the apical surface of RPE cells (R). The levels of all three proteins increase over time, and the immunolabeling pattern becomes more distinct at P10 and P15. All photomicrographs were taken at the same magnification. In the scale bar, X=25 μm.

Figure 5

Colocalization of CD81 with Sap97 and EBP50 at P20. The staining of the P20 rat retina for CD81 (A and D), Sap97 (B), and EBP50 (E) is shown in two double-stained sections. The merged images of these two sections are shown in C and F. The pattern of CD81 labeling is consistent with the labeling of Müller glial cells, as shown by the prominent labeling of the external limiting membrane (arrow, A) and retinal pigment epithelium (RPE) cells (double arrow, A). Sap97 shows a general stain in the retinal neurons and a prominent band at the base of the RPE (arrow in B). In the merged image (C), Sap97 immunoreactivity is colocalized with CD81 on the basolateral surface of RPE cells. EBP50 labels a prominent band at the junction between the retina and the PRE cells (arrow, E). The EBP50 immunoreactivity colocalizes with CD81 immunoreactivity at the apical surface of the RPE cells (F). In A and F, the layers of the P20 retina are shown: the ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), and outer nuclear layer (ONL). All photomicrographs are taken at the same magnification. The scale bar in F represents 25 μm.

Figure 6

Colocalization of CD81 with Sap97 and EBP50 at P2. The staining pattern of CD81, Sap97, and EBP50 in the developing rat retina is shown at P2. A and D are stained for CD81, B is stained for Sap97, and C is the merged image. E is stained for EBP50; F is the merged image. At this age, the ganglion cell layer (GCL) and inner plexiform layer (IPL) are present. The remainder of the retina consists of a neuroblastic layer (NBL). The arrows in B and E indicate the location of the developing retinal pigment epithelium (RPE) layer. All photomicrographs were taken at the same magnification. The scale bar in represents 25 μm.