PRG3 and PRG5 C-Termini: Important Players in Early Neuronal Differentiation

Abstract

The functional importance of neuronal differentiation of the transmembrane proteins' plasticity-related genes 3 (PRG3) and 5 (PRG5) has been shown. Although their sequence is closely related, they promote different morphological changes in neurons. PRG3 was shown to promote neuritogenesis in primary neurons; PRG5 contributes to spine induction in immature neurons and the regulation of spine density and morphology in mature neurons. Both exhibit intracellularly located C-termini of less than 50 amino acids. Varying C-termini suggested that these domains shape neuronal morphology differently. We generated mutant EGFP-fusion proteins in which the C-termini were either swapped between PRG3 and PRG5, deleted, or fused to another family member, plasticity-related gene 4 (PRG4), that was recently shown to be expressed in different brain regions. We subsequently analyzed the influence of overexpression in immature neurons. Our results point to a critical role of the PRG3 and PRG5 C-termini in shaping early neuronal morphology. However, the results suggest that the C-terminus alone might not be sufficient for promoting the morphological effects induced by PRG3 and PRG5.

Keywords: PRG3/LPPR1; PRG4/LPPR2; PRG5/LPPR5; hippocampal neurons; neuronal outgrowth.

Figure 1

PRG3 and PRG5 C-terminal domains are essential for inducing morphological changes in non-neuronal cells (A) Left panel: Schematic depiction of PRG3. Transmembrane domains TM1-TM6 are highlighted in light blue and the C- and N-terminal regions are located on the intracellular side. Single circles represent amino acids (aa) (one letter code). Right upper panel: Confocal stacks of representative HEK293H cells transfected with PRG3-EGFP fusion-encoding plasmids and stained for GFP. Arrows point to filopodia identified by F-actin staining (red, phalloidin). Arrowheads denote areas showing membrane localization. This refers also to (C). PM = plasma membrane. Right lower panel: Higher magnification of representative HEK293H cells transfected as indicated above and shown by white rectangles. (B) Left panel: Schematic depiction of PRG3∆C (43 aa deleted). Upper right panel: Confocal stacks of representative HEK293H cells transfected with PRG3∆C-encoding plasmids and stained for GFP (green fluorescent protein). Lower right panel: Higher magnification of representative HEK293H cells transfected as indicated above and shown by white rectangles. (C) Left panel: Schematic depiction of PRG3-C5. The sequence of exchanged C-terminal domain residues of PRG5 is shown in orange (aa 252–316). Upper right panel: Confocal stacks of representative HEK293H cells transfected with PRG3-C5-encoding plasmids and stained for GFP. Lower right panel: Higher magnification of representative HEK293H cells transfected as above and indicated by white rectangles. Cell nuclei were stained with DAPI (blue). The scale bar represents 10 µm in the upper panels and 5 µm in the lower panels.

Figure 2

PRG5 and PRG3 C-terminal domains are essential for inducing morphological changes in non-neuronal cells (A) Left panel: Schematic depiction of PRG5. Transmembrane domains TM1-TM6 are highlighted in light orange and the C- and N-terminal regions are located on the intracellular side. Single circles represent aa (one letter code). Right upper panel: Confocal stacks of representative HEK293H cells transfected with PRG5-encoding plasmids and stained for GFP. Arrows point to filopodia identified by F-actin staining, (red, phalloidin), while arrowheads denote areas showing membrane localization. This refers also to (B and C). PM = plasma membrane. Lower right panel: Higher magnification of representative HEK293H cells transfected as above and indicated by white rectangles. (B) Left panel: Schematic depiction of PRG5∆C (42 aa deleted). Upper right panel: Confocal stacks of representative HEK293H cells transfected with PRG5∆C-encoding plasmids and stained for GFP. PRG5∆C did not locate all filopodia as denoted by asterisks in the lower panel. Lower right panel: Higher magnification of representative HEK293H cells transfected as above and indicated by white rectangles. (C) Left panel: Schematic depiction of PRG5-C3. The sequence of exchanged C-terminal domain residues of PRG3 is shown in blue (aa 260–325). Upper right panel: Confocal stacks of representative HEK293H cells transfected with PRG5-C3-encoding plasmids and stained for GFP. Lower right panel: Higher magnification of representative HEK293H cells transfected as above and indicated by white rectangles. Cell nuclei were stained with DAPI (blue). The scale bar represents 10 µm in the upper panels and 5 µm in the lower panels.

Figure 3

The PRG3 C-terminal domain is an important player in the early development of hippocampal neurons. Subcellular distribution of PRG3-, PRG3∆C- and PRG3-C5-encoding fusion proteins in immature primary hippocampal neurons in culture. Hippocampal neurons in culture were transfected at DIV1 with (A) either PRG3- (upper panel), PRG3∆C- (middle panel), or PRG3-C5-encoding plasmids (lower panel), and analyzed at DIV2. Confocal stacks of representative neurons with immunocytochemistry of the respective fusion proteins and stained for GFP to visualize the morphology of the cell or tubulin are shown. Arrowheads denote membrane localization. The scale bar represents 10 µm. Boxplots showing the quantification of (B) the total number of neurites (*** p ≤ 0.0001); (C) the major neurite length (*** p ≤ 0.0001); (D) the number of branching points (*** p ≤ 0.0001) in PRG3-, PRG3∆C- and PRG3-C5-overexpressing neurons. Quantification of membrane protrusions in subgroups based on the length of the (E) group of short protrusions (0.2–<2 µm), (F) medium protrusions (2–<5 µm) and (G) long protrusions (5–<10 µm) (*** p ≤ 0.0001, ** p ≤ 0.01) in the same neurons (PRG3 n = 40, PRG3∆C n = 41, PRG3-C5 n = 38 neurons). All data are presented as median and IQR. Boxes range from 1st to 3rd quartile. Horizontal lines represent medians. Whiskers represent minimum and maximum. Dots represent the values of individual neurons. Statistical analysis was performed using the Kruskal–Wallis test. A level of confidence of p ≤ 0.05 was adopted ** p ≤ 0.01, *** p ≤ 0.001). PRG3 was always set to 100 and the ratios of changes of the different analyses of morphology are shown (B–G).

Figure 4

The PRG5 C-terminal domain is an important player in the early development of hippocampal neurons. Subcellular distribution of PRG5-, PRG5∆C- and PRG5-C3-encoding fusion proteins in immature, primary hippocampal neurons in culture. Hippocampal neurons in culture were transfected at DIV1 with (A) either PRG5- (upper panel), PRG5∆C- (middle panel), or PRG5-C3-encoding plasmids (lower panel), and analyzed at DIV2. Confocal stacks of representative neurons with immunocytochemistry of the respective fusion proteins stained for GFP to visualize the morphology of the cell or tubulin are shown. Arrowheads denote areas showing membrane localization. The scale bar represents 10 µm. Boxplots showing the quantification of (B) the total number of neurites; (C) the major neurite length (* p ≤ 0.01); (D) the number of branching points (** p ≤ 0.01) in PRG5-, PRG5∆C- and PRG5-C3-overexpressing neurons. Quantification of membrane protrusions in subgroups based on length in the (E) group of short protrusions (0.2–<2 µm), (F) medium protrusions (2–<5 µm) and (G) long protrusions (5–<10 µm). (PRG5 n = 44, PRG5∆C n = 37, PRG5-C3 n = 41 neurons; *** p ≤ 0.0001). All data are presented as median and IQR. Boxes range from 1st to 3rd quartile. Horizontal lines represent medians. Whiskers represent minimum and maximum. Dots represent the values of individual neurons. Statistical analysis was performed using the Kruskal–Wallis test. A level of confidence of p ≤ 0.05 was adopted (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). PRG5 was always set to 100 and the ratios of changes of the different morphology analyses are shown (B–G).

Figure 5

PRG3 and PRG5 C-terminal domains induce morphological changes when fused to PRG4 in non-neuronal cells. (A) Left panel: Schematic depiction of PRG4. Transmembrane domains TM1-TM6 are highlighted in light yellow, and the C- and N-terminal regions are located on the intracellular side. Single circles represent aa (one letter code). Upper right panel: Confocal stacks of representative HEK293H cells transfected with PRG4-encoding plasmids and stained for FLAG. Arrowheads point to filopodia identified by F-actin staining (red, phalloidin). This refers also to (B) and (C). PM = plasma membrane. Lower right panel: Higher magnification of representative HEK293H cells transfected as above and indicated by white rectangles. (B) Left panel: Schematic depiction of PRG4-C3. The sequence of exchanged C-terminal domain residues of PRG3 is shown in blue (aa 257–325; including TM6). Right upper panel: Confocal stacks of representative HEK293H cells transfected with PRG4-C3-encoding plasmids and stained for GFP. Lower right panel: Higher magnification of representative HEK293H cells transfected as above and indicated by white rectangles. (C) Left panel: Schematic depiction of PRG4-C5. The sequence of exchanged C-terminal domain residues of PRG5 is shown in orange (aa 252–316; including TM6). Upper right panel: Confocal stacks of representative HEK293H cells transfected with PRG4-C5-encoding plasmids and stained for GFP. Lower right panel: Higher magnification of representative HEK293H cells transfected as above and indicated by white rectangles. Cell nuclei were stained with DAPI (blue). The scale bar represents 10 µm in the upper panels and 5 µm in the lower panels.

Figure 6

The PRG3 C-terminal domain is crucial for neurite growth and membrane protrusions, but is not capable of restoring the full function of PRG3 when fused to PRG4. Subcellular distribution of PRG4- and PRG4-C3-encoding fusion proteins in immature primary hippocampal neurons in culture. Hippocampal neurons in culture were transfected at DIV1 with (A) either PRG4- (upper panel) or PRG4-C3-encoding plasmids (lower panel) and analyzed at DIV2. Confocal stacks of representative neurons with immunocytochemistry of the respective fusion proteins stained for FLAG (green, PRG4) or GFP (green, PRG4-C3) to visualize the morphology of the cell or tubulin are shown. Arrowheads denote membrane localization. The scale bar represents 10 µm. Boxplot showing the quantification of (B) the total number of neurites; (C) the quantification of major neurite length and (D) the number of branching points in PRG4-, PRG3- and PRG4-C3-overexpressing neurons. Quantification of membrane protrusions in subgroups based on length in the (E) group of short protrusions (0.2–<2 µm) (* p ≤ 0.01) and in subgroups of (F) medium protrusions (2–<5 µm) and (G) long protrusions (5–<10 µm) (* p ≤ 0.01, ** p ≤ 0.001). (PRG4 n = 30, PRG3 n = 30, PRG4-C3 n = 30 neurons). All data are presented as median and IQR. Boxes range from 1st to 3rd quartile. Horizontal lines represent medians. Whiskers represent minimum and maximum. Dots represent the values of individual neurons. Statistical analysis was performed using the Kruskal–Wallis test. A level of confidence of p ≤ 0.05 was adopted (* p ≤ 0.05, ** p ≤ 0.01,). PRG4 was always set to 100 and ratios of changes of the different morphology analyses are shown (B–G).

Figure 7

The PRG5 C-terminal domain is important in the early development of hippocampal neurons, but not sufficient to restore the full function of PRG5 when fused to PRG4. Subcellular distribution of PRG4- and PRG4-C5-encoding fusion proteins in immature primary hippocampal neurons in culture (note that the images of PRG4 are the same as in Figure 6A for better comparison). Hippocampal neurons in culture were transfected at DIV1 with (A) either PRG4- (upper panel) or PRG4-C5-encoding plasmids (lower panel) and analyzed at DIV2. Confocal stacks of representative neurons with immunocytochemistry of the respective fusion proteins stained for FLAG (green, PRG4) or GFP (green, PRG4-C5) to visualize the morphology of the cell or tubulin are shown. Arrowheads denote areas showing membrane localization. The scale bar represents 10 µm. Boxplots showing the quantification of (B) the total number of neurites (*** p ≤ 0.0001, * p ≤ 0.01); (C) the quantification of major neurite length (* p ≤ 0.01), and (D) the number of branching points (*** p ≤ 0.001) in PRG4- PRG5- and PRG4-C5-overexpressing neurons. Quantification of membrane protrusions in subgroups based on length in the (E) group of short protrusions (0.2–<2 µm) (*** p ≤ 0.0001; ** p ≤ 0.001) and of (F) medium protrusions (2–<5 µm) and (G) long protrusions (5–<10 µm) (*** p ≤ 0.0001). (PRG4 n = 30, PRG5 n = 30, PRG4-C5 n = 30 neurons). All data are presented as median and IQR. Boxes range from 1st to 3rd quartile. Horizontal lines represent medians. Whiskers represent minimum and maximum. Dots represent the values of individual neurons. Statistical analysis was performed using the Kruskal–Wallis test. A level of confidence of p ≤ 0.05 was adopted (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). PRG4 was always set to 100 and ratios of changes of the different morphology analyses are shown (B–G).