L-DOPA is an endogenous ligand for OA1

Abstract

Albinism is a genetic defect characterized by a loss of pigmentation. The neurosensory retina, which is not pigmented, exhibits pathologic changes secondary to the loss of pigmentation in the retina pigment epithelium (RPE). How the loss of pigmentation in the RPE causes developmental defects in the adjacent neurosensory retina has not been determined, but offers a unique opportunity to investigate the interactions between these two important tissues. One of the genes that causes albinism encodes for an orphan GPCR (OA1) expressed only in pigmented cells, including the RPE. We investigated the function and signaling of OA1 in RPE and transfected cell lines. Our results indicate that OA1 is a selective L-DOPA receptor, with no measurable second messenger activity from two closely related compounds, tyrosine and dopamine. Radiolabeled ligand binding confirmed that OA1 exhibited a single, saturable binding site for L-DOPA. Dopamine competed with L-DOPA for the single OA1 binding site, suggesting it could function as an OA1 antagonist. OA1 response to L-DOPA was defined by several common measures of G-protein coupled receptor (GPCR) activation, including influx of intracellular calcium and recruitment of beta-arrestin. Further, inhibition of tyrosinase, the enzyme that makes L-DOPA, resulted in decreased PEDF secretion by RPE. Further, stimulation of OA1 in RPE with L-DOPA resulted in increased PEDF secretion. Taken together, our results illustrate an autocrine loop between OA1 and tyrosinase linked through L-DOPA, and this loop includes the secretion of at least one very potent retinal neurotrophic factor. OA1 is a selective L-DOPA receptor whose downstream effects govern spatial patterning of the developing retina. Our results suggest that the retinal consequences of albinism caused by changes in melanin synthetic machinery may be treated by L-DOPA supplementation.

Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1. Cell Surface OA1

(A–C) Western blot analysis of proteins bound (B) or unbound (U) to strepavidin-conjugated beads after biotinylation of RPE in situ, cultured RPE (B), or COS cells transfected to express OA1-GFP (C). Blots were probed to visualize OA1 and actin after cell surface biotinylation and fractionation using strepavidin-conjugated beads. For cultured cells (B and C), cells were either maintained in 500 μM (normal DMEM) or 1 μM tyrosine for 3 d prior to analysis. (D) Quantification of western blot analysis by densitometry. OA1 densitometry is shown as the percentage of the control for paired cell cultures, transfected, and then split into two equal groups, one of which was the control, maintained in normal DMEM (control; open bars). The other group was maintained in 1 μM tyrosine DMEM (LT; solid bars) until harvest. Paired t-test analysis was used to test whether the difference was significant, and an asterisk (*) denotes p < 0.001. Actin, analyzed the same way, showed no differences, and p = 0.724. (E and F) Composite confocal microscopy of pigmenting RPE cells maintained in normal DMEM (E) or 1 μM tyrosine (F), and then stained with anti-OA1 antibodies and imaged at 20×. Bar represents 25 μm.

Figure 2. Increased Intracellular Calcium in Response to L-DOPA.

(A) Representative traces of [Ca2+]i during the time course of the standard experimental protocol in transfected and untransfected CHO cells. After establishment of a stable baseline for 3 min, the test agent was added at 1 μM. At 5 min, KCl was added to serve as a control that the cells were Fura-2 loaded and patent. Identical protocols were performed for both transfected cells and paired untransfected cells. (B) Summary data for [Ca2+]i in response to tyrosine, dopamine, and L-DOPA in transfected and untransfected CHO cells. Untransfected cells are shown with L-DOPA treatment. Our experimental control of membrane depolarization with KCl is also shown. Each bar represents data collected from at least ten experiments and is presented as the mean change from baseline [Ca2+]i after test agent addition. Error bars represent SD, t-test analyses were used to test for significant differences, and an asterisk (*) denotes p < 0.01. (C) Analysis of pertussis toxin sensitivity of [Ca2+]i increase in cells transfected to express OA1 or RPE that express the natural protein. Data represent mean of at least six experiments for each group of transfected cells and 20 individual experiments for each of the treated and untreated RPE with endogenous OA1 expression. The t-test analyses were used to test for significant differences, and an asterisk (*) denotes p < 0.01. (D) cAMP was measured in CHO transfected to express OA1. The control group represents transfected, but untreated, CHO cells and the basal level of cAMP in those cells. Cells were treated with 1.0 μM L-DOPA, 0.1 μM forskolin, L-DOPA + 0.1 μM forskolin, and as a positive control, 1 μM forskolin. Results represent the mean cAMP levels observed in at least six experiments in which all experimental groups were analyzed in a paired fashion using replicate monolayers in the same culture plate. Error bars represent the SD of each group, and the only significant difference observed was the increase in cAMP levels after forskolin treatment.

Figure 3. OA1 Ligand Binding

(A) Binding kinetics between OA1 and L-DOPA were determined using radiolabeled ligand binding assays. Results represent data collected from five such experiments and are presented as mean specific binding ± SEM. The hyperbolic curve fit exhibited an r2 value of 0.994, Kd was determined to be 9.34 × 10−6 M ± 1.14 × 10−6 M. The inset Scatchard plot illustrates the kinetics of a single-site binding relationship. (B) We examined the comparative binding of 5 μM [H3] L-DOPA to OA1-transfected CHO cells in the presence of 1.0 mM dopamine, tyrosine, or L-DOPA. The data represent mean total binding ± SD for each group. An asterisk (*) denotes p < 0.05 when comparing the results between the control group to the binding in the presence of the potential competitive ligands. (C) Competitive interaction between 5 μM [H3] L-DOPA and dopamine was assessed to determine whether dopamine functions as an antagonist of OA1 activity. Results indicate that dopamine and L-DOPA compete for the same OA1 binding site, and the data fit the binding model with an r2 value of 0.95. The Ki for dopamine was 2.388 ± 0.266 μM (mean ± SEM), similar to the Kd for L-DOPA. (D) Dose-dependent OA1 signaling through OA1. Data represent mean increase in [Ca2+]i elicited by L-DOPA treatment of the cells at the concentrations given (n = 6 for each dose). We used t-test analyses to compare between the responses achieved at each dose, and an asterisk (*) denotes p < 0.01 for the comparison at 1 and 10 μM.

Figure 4. Ligand-Dependent Recruitment of β-arrestin to OA1

(A–F) All images represent 2-μm-thick confocal sections of CHO cells transfected to express OA1-GFP (green). β-arrestin was visualized using immunofluorescence methods (red) prior to addition of L-DOPA (A–C) and after treatment with 1 μM L-DOPA (D–F), and the merged images (C and F) illustrate regions where the two proteins colocalize, at the resolution of white light imaging. Bar represents 10 μm. (G and H) are low magnification of field images of transfected CHO cells, with two transfected cells visible (arrows) (G). The remainder of the cell population is visualized using antibodies to β-arrestin (H) to illustrate that β-arrestin recruitment to the membrane only occurred in the OA1-expressing cells (arrows). Bar represents 25 μm.

Figure 5. l-DOPA Stimulates PEDF Production in RPE

(A) PEDF concentrations were determined by ELISA of cell-conditioned medium. RPE cells were control cells that were without L-DOPA treatment (open bar), or OA1-stimulated cells that were treated with 1 μM L-DOPA prior to being maintained for 3 d in normal DMEM (solid bar). Data are presented as the mean of three experiments conducted in triplicate, error bars represent SD, and an asterisk (*) denotes p < 0.01 using a paired t-test. (B) PEDF concentrations in conditioned medium from pigmenting RPE determined by ELISA. Cells were either control pigmenting RPE cultures (open bar) or paired cultures treated with phenylthiourea (PTU) at 200 μM (solid bar). Data are presented as the mean of three experiments conducted in triplicate, error bars represent SD, and an asterisk (*) denotes p < 0.01 using a paired t-test. (C) PEDF concentrations in conditioned medium of pigmented RPE cells treated with PTU and then treated with L-DOPA to stimulate OA1 signaling. ELISA assays were conducted prior to PTU treatment (open bar), then after PTU treatment (solid bar), and then from the same cultures after L-DOPA stimulation (checkered bar). Results are presented as mean ± SD of the value achieved related to that culture of cells. An asterisk (*) denotes p < 0.01 when comparing PTU to the control (same culture tested prior to PTU), and L-DOPA/PTU compared to the PTU sample from that same culture.