Ligand-induced increase of PRLR localizations and decrease of GHR localizations at the surface of T47D cells. A representative set of reconstructed dSTORM images. GHR is labeled with Alexa 568-conjugated antibody (green, channel 1), and PRLR is labeled with Alexa 647-conjugated antibody (red, channel 2). The last column of images shows the merging of these two channels. T47D cells were either left untreated (upper row), exposed to 500ng/mL GH for 3 min (middle row), or exposed to 500ng/mL PRL for 3 min (lower row). Brightness was increased by 40% and Contrast was reduced by 40% to increase visibility. Scale bars 5μm.

(A, B) Quantification of GH-induced (500 ng/ml) (A) and PRL-induced (500 ng/ml) (B) changes of PRLR localizations on the cell surface. (C, D) Quantification of GH-induced (C) and PRL-induced (D) changes of GHR localizations on the cell surface. Each data point represents the density of receptors in a 6.25 μm X 6.25 μm area. Data are collected from at least 6 cells from each group and displayed as mean ± SE. * (P<0.05), ** (P<0.01), and *** (P<0.001) indicate the statistical significance in comparison with Basal and are calculated by two-tailed t-tests assuming unequal variances. (E) Model depicting hPRLR and hGHR densities and activation schemes in T47D cells.

GH/PRL stimulation induces a redistribution of hGHR and hPRLR clusters. (A) Sample dSTORM images and application of DBSCAN to identify and measure clusters (scale bar, 1 μm). (B) Left panels: Representative dSTORM images of hGHR (top) and hPRLR (bottom) on the T47D cell surface in resting state (scale bar, 5 μm). Brightness was increased by 40% for better visibility. Right panels: The corresponding histograms of the relative frequencies of localizations. (C, D) The medians of hGHR and hPRLR cluster sizes following GH (C) and PRL (D) treatment are summarized in these plots. The x-axis and y-axis represent hPRLR and hGHR cluster sizes, respectively. Data are collected from at least 6 cells from each group and displayed as median ± SE.

Spatial proximity of hGHR and hPRLR upon ligand stimulation. (A) Proximity ligation assays were performed in T47D cells (scale bar, 10 μm). Here we show representative confocal microscopy images of T47D cells at resting and ligand stimulation conditions. The PLA signal (red dots) corresponds to hGHR and hPRLR complexes, wheat germ agglutinin (green staining) corresponds to the cell membrane, and DAPI (blue staining) corresponds to cell nuclei. (B, C) hGHR and hPRLR colocalization changes in T47D cell surface following 500 ng/ml GH (B) or PRL (C) treatment. Each data point represents a ratio of clusters, which contain both hGHR and hPRLR, to the total clusters on the cell surface. Data are displayed as mean± SE. * (P<0.05), ** (P<0.01), and *** (P<0.001) indicate the statistical significance in comparison with Basal and are calculated by a two-tailed t-test assuming unequal variance.

PRL-induced reduction of hGHR on the cell surface depends on the presence of PRLR. (A) Detergent cell extracts of T47Dvec, T47DΔGHR, and T47DΔPRLR were analyzed by immunoblotting with anti-GHRcyt-AL47, anti-PRLRcyt, and anti-β-actin antibodies. (B) In T47DΔPRLR, GH (500 ng/ml) induces downregulation of hGHR localizations on the cell surface, while (C) PRL does not change hGHR localizations. (D, E) In T47DΔGHR cells, GH (D) and PRL (E) incite an increase of hPRLR on the cell surface. Data are displayed as mean ± SE. * (P<0.05), ** (P<0.01), and *** (P<0.001) indicate the statistical significance in comparison with Basal and are calculated by a two-tailed t-test assuming unequal variance. (F) γ2A-JAK2 cells with stable expression of hGHR or hPRLR. Detergent extracts were resolved by SDS-PAGE and immunoblotted with anti-GHRcyt- AL47 and anti-PRLRcyt-AL84 antibodies. (G, H) Serum-starved γ2A-JAK2-hGHR cells (G) or γ2A-JAK2-hPRLR cells (H) were treated with GH (500 ng/ml) or PRL (500 ng/ml) for 5 min. Localizations of hGHR in γ2A-JAK2-hGHR cells were decreased after GH treatment while remaining the same after PRL treatment compared to the basal state. Localizations of hPRLR in γ2A-JAK2-hPRLR cells were increased by GH or PRL treatment. Data are displayed as mean ± SE. * (P<0.05), ** (P<0.01), and *** (P<0.001) denote the statistical significance in comparison with Basal and are calculated by a two-tailed t-test assuming unequal variance.

Box 1 region in hPRLR contributes to PRLR-induced GHR downregulation. (A) Diagrams of employed mutant hPRLR variants with truncations or deletion in the intracellular domain. ECD, extracellular domain; TMD, transmembrane domain; ICD, intracellular domain. (B) γ2A-JAK2-hGHR cells were transiently transfected with hPRLR-tr292 and hPRLR-tr238. Cell extracts were resolved by SDS-PAGE and blotted with anti-PRLR mAbext-1.48 and anti-PRLRcyt-AL84. (C-G) γ2A-JAK2-hGHR cells were transfected with (C) wild-type hPRLR (hPRLR-WT), (D) hPRLR truncated at 292 aa (hPRLR-tr292), (E) hPRLR truncated at 238 aa (hPRLR-tr238), (F) hPRLR with box1 motif deleted (hPRLR-ΔBox1) and (G) vector pcDNA3.1 (vector). Transfected cells were imaged by dSTORM microscopy and analyzed using the DBSCAN algorithm. The density of hGHR localizations on the cell surface was calculated. Each data point represents the density of hGHR in a cell surface area of size 6.25 μm X 6.25 μm. Data are collected from at least 6 cells (4 ROIs per cell) from each group, and values are displayed as mean ± SE (from three independent experiments). Data are normalized such that the basal is 100%. * (P<0.05), ** (P<0.01), and *** (P<0.001) denote the statistical significance in comparison with Basal and are calculated by a two-tailed t-test assuming unequal variance. (H) Detergent cell extracts of hPRLR-ΔBox1 and hGHR-expressing cells were analyzed by immunoblotting. After 5 hrs. starvation, cells were treated with GH (500 ng/ml) or PRL (500 ng/ml) for 10 min. In each experiment, the average Basal value is considered 100%. (I) Fraction of GHR/PRLR colocalized clusters. In the resting state, the colocalization ratio is significantly lower for cells expressing hPRLR-tr238 than for those expressing hPRLR-WT. Each data point represents the ratio of the number of clusters, which contain both hGHR and hPRLR, to the total number of clusters on the cell surface. Data are displayed as mean ± SE. * (P<0.05) indicates statistical significance in comparison with WT and is calculated by a two-tailed t-test assuming unequal variance (all bars without an asterisk are not significant).

Box 1 region in hGHR also plays an important role in PRLR and GHR interaction. (A) Diagram of hGHR-ΔBox1 showing the deleted box 1 region in the intracellular domain of hGHR. (B) γ2A-JAK2-hPRLR cells were transfected with hGHR-ΔBox1. Transfected cells were imaged by dSTORM microscopy and analyzed using DBSCAN. The density of hGHR localizations on the cell surface was calculated. Each data point represents the density of hGHR in a cell surface area of size 6.25 μm X 6.25 μm. Data are collected from at least 6 cells for each group and displayed as mean ± SE. * (P<0.05), ** (P<0.01), and ***(P<0.001) indicate the statistical significance in comparison with Basal and are calculated by a two-tailed t-test assuming unequal variance.

Model of JAK2 stabilizing GHR-GHR and PRLR-PRLR heteromers. (A) Schematic representation of GHRs homodimer and PRLRs homodimer form a heteromer, which contains two PRLRs (red), two GHRs(green), and four JAK2(blue). (B) A top view of the JAK2/PRLR/GHR multimer.

The size distributions of co-localized clusters are shown in 3D plots, where the binned x-axis represents the number of hGHR localizations, the binned y-axis represents the number of hPRLR localizations, and the z-axis represents the probability of the cluster. Each bin has a size of 200 localizations. Both GH (A) and PRL (B) induce a distinct change in the composition of co-localized clusters.