Combinatorial expression of alpha- and gamma-protocadherins alters their presenilin-dependent processing

Abstract

Alpha- and gamma-protocadherins (Pcdhs) are type I transmembrane receptors expressed predominantly in the central nervous system and located in part in synapses. They are transcribed from complex genomic loci, giving rise in the mouse to 14 alpha-Pcdh and 22 gamma-Pcdh isoforms consisting of variable domains, each encompassing the extracellular region, the transmembrane region, and part of the intracellular region harboring the alpha- or gamma-Pcdh-specific invariant cytoplasmic domain. Presenilin-dependent intramembrane proteolysis (PS-IP) of gamma-Pcdhs and the formation of alpha/gamma-Pcdh heteromers led us to investigate the effects of homo- and heteromer formation on gamma- and putative alpha-Pcdh membrane processing and signaling. We find that upon surface delivery, alpha-Pcdhs, like gamma-Pcdhs, are subject to matrix metallo-protease cleavage followed by PS-IP in neurons. We further demonstrate that the combinatorial expression of alpha- and gamma-Pcdhs modulates the extent of their PS-IP, indicating the formation of alpha/gamma-Pcdh heteromers with an altered susceptibility to processing. Cell-specific expression of alpha/gamma-Pcdh isoforms could thus determine cell and synapse adhesive properties as well as intracellular and nuclear signaling by their soluble cytoplasmic cleavage products, alpha C-terminal fragment 2 (alpha-CTF-2) and gamma-CTF-2.

FIG. 1.

Expression and processing of Pcdhα4 and PcdhγAI in SH-SY5Y cells. (Α) Schematic representation of γ-Pcdh PS-IP. The variable region of α- and γ-Pcdhs consists of six extracellular cadherin repeats (EC repeats), a transmembrane region, and a short intracellular part that is connected to the constant region (CR). The main features of γ-Pcdh PS-IP are an MMP cleavage (blocked by TAPI-1) that generates a soluble ECD and a membrane stump (CTF-1; ∼30 kDa) (molecular mass markers [in kilodaltons] are shown on the right), followed by γ-secretase (γ-Sec.) cleavage of the CTF-1 (blocked by DAPT), releasing the intracellular domain (CTF-2; ∼26 kDa). (B) Expression scheme for tagged Pcdhs and immunoblots. Middle panel, immunoblot with anti-Cα Ab (Cα) of whole-cell lysates of SH-SY5Y cells transiently expressing myc-PcdhγAI (lane 1) or CFP-Pcdhα4 (lane 2). Arrows indicate the immunoreactive bands detected with Cα Ab for CFP-Pcdhα4 (131 kDa; CFP-α4) and α4-CTF-1 (∼30 kDa). Upper panel, immunoblot with anti-GFP Ab (GFP) of corresponding cell culture medium. The arrow indicates the immunoreactive band for the shedded ectodomain of CFP-Pcdhα4 (∼100 kDa; α4-ECD). Lower panel, immunoblot of whole-cell lysates with anti-β-actin Ab (β-actin) serving as the loading control. (C) Left upper panel, immunoblot with anti-Cγ Ab (Cγ) of whole-cell lysates of SH-SY5Y cells transiently expressing myc-PcdhγAI (lane 1) or CFP-Pcdhα4 (lane 2). Arrows indicate the immunoreactive bands for myc-PcdhγAI (131 kDa; myc-γAI) and γAI-CTF-1 (∼30 kDa). Left lower panel, immunoblot of whole-cell lysates with anti-β-actin Ab (β-actin) serving as the loading control. Above the immunoblots is the scheme for the expression of tagged Pcdhs in SH-SY5Y cells. Right upper panel, immunoblot with anti-Cγ Ab (Cγ) of murine Pcdhγ^+/+ (lane 3) or Pcdhγ^−/− (lane 4) whole-brain extracts. Right lower panel, immunoblot of corresponding lysates with anti-β-actin Ab (β-actin) serving as the loading control. A scheme representing the genotypes of the mice used for whole-brain protein lysates is shown above the immunoblots.

FIG. 2.

Sequential MMP and γ-secretase cleavage of CFP-Pcdhα4 in SH-SY5Y cells. (A) Upper panel, immunoblot with anti-Cα Ab (Cα) of whole-cell lysates of SH-SY5Y cells transiently expressing CFP-Pcdhα4 and treated with DMSO (lane 1), TAPI-1(lane 2), or DAPT (lane 3). Arrows indicate the immunoreactive bands for CFP-Pcdhα4 (131 kDa; CFP-α4) (molecular mass markers [in kilodaltons] are shown on the right) and α4-CTF-1 (∼30 kDa). Lower panel, immunoblot of the lysates with anti-β-actin Ab (β-actin) serving as the loading control. Above the immunoblots is a scheme for the expression of CFP-Pcdhα4 and treatment with TAPI-1 or DAPT in SH-SY5Y cells. (B) Quantification of results shown in panel A for α4-CTF-1 levels compared to β-actin and, as a positive control, for γAI-CTF-1 levels in transiently YFP-PcdhγAI-expressing SH-SY5Y cells, treated with DMSO, TAPI-1, or DAPT (as percentages of the control; n = 4). The bar graph includes the average values ± standard errors of the means (SEM), and significant differences (**, P ≤ 0.01 by ANOVA) between control (DMSO) and treatment (TAPI-1 or DAPT) conditions are indicated. (C) Immunoblot with anti-Cα Ab (Cα) of SH-SY5Y and presenilin dko cells transiently expressing CFP-Pcdhα4 (lanes 2 to 4), treated with either DMSO (lane 2) or lactacystin (lanes 1, 3, and 4). Arrows indicate the immunoreactive band for CFP-Pcdhα4 (CFP-α4) and the corresponding α4-CTF-1 and α4-CTF-2 (∼26-kDa) bands. The scheme above the immunoblots shows the expression of CFP-Pcdhα4 and lactacystin treatment of SH-SY5Y cells. (D) Upper panel, immunoblot with anti-Cα Ab (Cα) of SH-SY5Y cells transiently expressing CFP-Pcdhα4 (lanes 1 to 3) and treated with lactacystin (lanes 1 to 3) and either DAPT (lane 2) or TAPI-1 (lane 3). Lower panel, immunoblot of corresponding lysates with anti-β-actin Ab (β-actin) serving as the loading control. Above the immunoblots is a scheme representing the expression of CFP-Pcdhα4 and lactacystin, DAPT, or TAPI-1 treatment of SH-SY5Y cells.

FIG. 3.

ADAM10 cleavage of Pcdhα4. (A) Immunoblot with anti-ADAM10, -15, and -17 and anti-Cα and -β-actin Ab of whole-cell protein lysate of SH-SY5Y cells transiently expressing CFP-Pcdhα4 and cotransfected with different siRNAs (lane 1, si control; lane 2, si-ADAM10; lane 3, si-ADAM15; lane 4, si-ADAM17). Arrows indicate the immunoreactive bands for CFP-Pcdhα4 (CFP-α4; 131 kDa) (molecular mass markers [in kilodaltons] are shown on the right) and its corresponding α4-CTF-1 (∼30 kDa). (B) Quantification of results shown in panel A. The bar graph includes the average values ± SEM, and significant differences (**, P ≤ 0.01 by ANOVA) for quantifying changes in α4-CTF-1 levels compared to β-actin (as percentages of the control; n = 3) are indicated.

FIG. 4.

Effects of Pcdhα4 on PcdhγAI cleavage in SH-SY5Y cells. (A and B) Surface immunocytochemistry with anti-GFP Ab (red) of SH-SY5Y cells transiently expressing YFP-PcdhγAI (A) or coexpressing YFP-PcdhγAI/myc-Pcdhα4 (B) (scale bar = 10 μm). Direct YFP fluorescence is shown in green (YFP). (C) Upper panel, immunoblot with anti-Cγ Ab (Cγ) of protein lysates of SH-SY5Y cells transiently expressing either YFP-PcdhγAI (lane 1) or coexpressing YFP-PcdhγAI/myc-Pcdhα4 (lane 2). Arrows mark immunoreactive bands corresponding to YFP-PcdhγAI (YFP-γAI; ∼130 kDa) (molecular mass markers [in kilodaltons] are shown on the right) and γAI-CTF-1 (∼30 kDa). Lower panel, immunoblot of corresponding lysates with anti-β-actin Ab (β-actin) serving as the loading control. The scheme above the immunoblots shows the expression of YFP-PcdhγAI and myc-Pcdhα4 in SH-SY5Y cells. (D) Quantification of results shown in panel C. The bar graph includes the average values ± SEM, and significant differences (**, P ≤ 0.01 by t test) quantifying changes in γAI-CTF-1 and YFP-γAI levels compared to β-actin (as percentages of the amounts in YFP-PcdhγAI-expressing cells; n = 4) are indicated. (E and F) Surface immunocytochemistry with anti-GFP Ab (red) of SH-SY5Y cells transiently expressing CFP-Pcdhα4 (E) or coexpressing CFP-Pcdhα4/myc-PcdhγAI (F). Direct CFP fluorescence is shown in green (CFP). (G) Upper panel, immunoblot with anti-Cα Ab (Cα) of protein lysates of SH-SY5Y cells transiently expressing either CFP-Pcdhα4 (lane 1) or coexpressing CFP-Pcdhα4/myc-PcdhγAI (lane 2). Arrows mark immunoreactive bands corresponding to CFP-Pcdhα4 (CFP-α4; ∼130 kDa) and α4-CTF-1 (∼30 kDa). Lower panel, immunoblot of corresponding lysates with anti-β-actin Ab (β-actin) serving as the loading control. Above the immunoblots is a scheme representing the expression of CFP-Pcdhα4 and myc-PcdhγAI in SH-SY5Y cells. (H) Quantification of results shown in panel G. The bar graph includes the average values ± SEM, and significant differences (**, P ≤ 0.01 by t test) quantifying changes in α4-CTF-1 and CFP-α4 levels compared to β-actin (as percentages of the amounts in CFP-Pcdhα4-expressing cells; n = 4) are indicated.

FIG. 5.

MMP and γ-secretase cleavage of myc-Pcdhα4 in primary neurons. (A) Colocalization of overexpressed myc-Pcdhα4 and endogenous, cellular γ-Pcdh. Upper panels, surface immunocytochemistry with anti-myc Ab (myc; red) and cellular immunocytochemistry with anti-Cγ Ab (Cγ; green) of DIV 21 primary hippocampal cultures infected with rAAV expressing myc-Pcdhα4 (scale bar = 10 μm). Colocalization of surface myc-Pcdhα4 and cellular γ-Pcdh is shown in yellow (merge). Lower panels, higher-magnification pictures of the insets in the upper panels (scale bar = 3 μm; arrows indicate selected yellow puncta). (B) Left upper panel, immunoblot analysis with anti-Cα Ab (Cα) of protein lysates of DIV 14 rat primary cortical cultures, either uninfected (control neurons, lane 1) or infected with rAAV expressing myc-Pcdhα4 (lane 2). Right upper panel, immunoblot analysis with anti-Cγ Ab of rat cortical neurons, either uninfected (control neurons, lane 3) or infected with rAAV expressing myc-PcdhγAI (lane 4). A molecular mass marker (in kilodaltons) is shown between left and right upper panels. Right and left lower panels, immunoblots of corresponding lysates with anti-β-actin Ab (β-actin) serving as the loading controls. The scheme above the immunoblots represents the expression of myc-Pcdhα4 and myc-PcdhγAI in neurons. (C) Quantification of results shown in panel B. The bar graph includes the average values ± SEM, and significant differences (**, P ≤ 0.01 by t test) for full-length α- and γ-Pcdhs between uninfected and infected neurons (as percentages of endogenous α/γ-Pcdh levels; n = 4) are indicated. (D) Upper panel, immunoblot analysis with anti-Cα Ab (Cα) of DIV 14 primary cortical cultures infected with rAAV expressing myc-Pcdhα4. For treatments, see the scheme above the immunoblot. Arrows indicate the immunoreactive bands for myc-Pcdhα4 (myc-α4; 100 kDa) and its corresponding α4-CTF-1 and α4-CTF-2 (∼26 kDa). Lower panel, immunoblot of corresponding lysates with anti-β-actin Ab (β-actin) serving as the loading control.

FIG. 6.

Surface expression of myc-Pcdhα4 in γ-Pcdh^+/+, γ-Pcdh^+/−, and γ-Pcdh^−/− neurons. (A) Upper panels, surface immunocytochemistry with anti-myc Ab of DIV 21 γ-Pcdh^+/+, γ-Pcdh^+/−, or γ-Pcdh^−/− murine primary cortical cultures infected with rAAV expressing myc-Pcdhα4 (scale bar = 10 μm). Lower panels, higher magnification of the insets in the upper panels (scale bar = 3 μm). (B) Quantification of results shown in panel A. Bar graph of the surface mean intensities, mean areas, and mean numbers of puncta of myc-Pcdhα4 in γ-Pcdh^+/+, γ-Pcdh^+/−, or γ-Pcdh^−/− neurons (as percentages of results for γ-Pcdh^+/+; dendrites of 12 different cells from three replicate experiments; n = 12). (C and D) Surface biotinylation of γ-Pcdh^+/+, γ-Pcdh^+/−, and γ-Pcdh^−/− neurons. (C) Immunoblot analysis with anti-Cα, -Cγ, -NR1, and -β-actin Ab (Cα, Cγ, NR1, and βactin) of protein lysates of DIV 14 surface-biotinylated murine γ-Pcdh^+/+, γ-Pcdh^+/−, and γ-Pcdh^−/− cortical primary cultures. Left panel (lanes 1 to 3), crude whole-cell lysate used as input for the IPr. Right panel (lanes 4 to 6), IPr of the surface-biotinylated proteins. (D) Quantification of results shown in panel C. Bar graph of α- and γ-Pcdh surface delivery normalized to NR1 surface delivery (as percentages of results for γ-Pcdh^+/+; n = 4). (E) Immunoblot analysis with anti-Cγ (Cγ) and anti-Cα (Cα) Ab of forebrain cellular (left panel) and PSD (right panel) protein fractions of P0 γ-Pcdh^+/+ and γ-Pcdh^−/− mice.

FIG. 7.

α-siRNA and surface expression of myc-PcdhγAI in neurons. (A) Upper panel, immunoblot analysis with anti-Cα Ab of protein lysates of DIV14 murine primary cortical cultures infected with lentivirus expressing either GFP-siRNA (cont.-siRNA; lane 1) or α-siRNA (lane 2). Arrows indicate the immunoreactive bands for endogenous full-length α-Pcdh (Pcdhα) and α-CTF-1 as detected by Cα Ab (Cα) (molecular mass markers [in kilodaltons] are shown on the right). Middle panel, immunoblot analysis of full-length γ-Pcdh (Pcdhγ) with anti-Cγ Ab (Cγ) of lysates from panel A. Lower panel, immunoblot with β-actin Ab (β-actin) serving as the loading control. (B and C) Upper panels, surface immunocytochemistry with anti-myc Ab (myc) of murine primary cortical culture coinfected with rAAV expressing myc-PcdhγAI and lentivirus expressing either control siRNA (B) or α-siRNA (C) (scale bar = 10 μm). Direct GFP fluorescence is shown in green. Lower panels, higher magnification of the insets in the upper panels (scale bar = 2 μm). (D) Quantification of results shown in panel C. Bar graphs show the surface mean intensities, mean areas, and mean numbers of puncta of myc-PcdhγAI in neurons coexpressing myc-PcdhγAI and either control siRNA or α-siRNA ± SEM (as percentages of the amount in myc-PcdhγAI/control siRNA-expressing neurons; dendrites of seven different cells from three replicate experiments; n = 7).

FIG. 8.

Effects of myc-Pcdhα4 on myc-PcdhγAI cleavage in neurons. (A) Upper panel, immunoblot analysis with anti-Cγ Ab (Cγ) of protein lysates of DIV 14 rat cortical cultures double-infected with rAAVs expressing either myc-PcdhγAI/GFP (lane 1) or myc-PcdhγAI/myc-Pcdhα4 (lane 2) (molecular mass markers [in kilodaltons] are shown on the right). Lower panel, immunoblot of corresponding lysates with anti-β-actin Ab (β-actin) serving as the loading control. Above the immunoblots is a scheme representing the expression of myc-Pcdhα4, myc-PcdhγAI, or GFP in neurons. (B) Quantification of results shown in panel A. The bar graph includes the average values ± SEM, and significant differences (**, P ≤ 0.01 by t test) for γAI-CTF-1 and myc-γAI in myc-PcdhγAI/GFP or myc-PcdhγAI/myc-Pcdhα4-expressing, double-infected, primary cortical cultures (as percentages of the amount in myc-PcdhγAI/GFP-expressing neurons; n = 5) are indicated. (C) Upper panel, immunoblot with anti-N-cadherin Ab (N-Cad.) of protein lysates of DIV 14 rat cortical cultures double-infected with rAAVs expressing either myc-PcdhγAI/GFP (lane 1) or myc-PcdhγAI/myc-Pcdhα4 (lanes 2 and 3). To better visualize the N-cadherin CTF-1 (N-Cad/CTF-1), cultures were treated with DAPT (lanes 1 and 2) or, as the control, with DMSO (lane 3). Lower panel, immunoblot of corresponding lysates with anti-β-actin Ab (β-actin) serving as the loading control. Above the immunoblot is a scheme representing the expression of myc-Pcdhα4, myc-PcdhγAI, GFP, and DAPT treatment in neurons. (D) Quantification of results shown in panel C. The bar graph includes the average values ± SEM, and significant differences (**, P ≤ 0.01 by t test) for the C-terminal N-cadherin cleavage product (N-Cad/CTF-1) and full-length N-cadherin (N-Cad/FL) in myc-PcdhγAI/GFP- or myc-PcdhγAI/myc-Pcdhα4-expressing, double infected, primary cortical cultures treated with DAPT (as percentages of the amount in myc-Pcdhα4/GFP-expressing neurons; n = 5) are indicated.

FIG. 9.

Nuclear and cytoplasmic localization of the α-CD. (A) Immunoblot analysis with anti-Cα Ab (Cα) of whole-cell protein lysates of SH-SY5Y cells transiently expressing CFP-Pcdhα4 and treated with lactacystin (lane 1) or expressing myc-α-CD and treated with DMSO (lane 2). Above the immunoblot is a scheme representing the expression of CFP-Pcdhα4, α-CD, and lactacystin treatment of SH-SY5Y cells. (B) Immunocytochemistry with anti-Cα Ab (Cα; red) and DAPI (blue) of SH-SY5Y cells transiently expressing myc-α-CD. Colocalization of DAPI and Cα fluorescence is shown in pink (merge). (C) Immunocytochemistry with anti-myc Ab (myc; red) and DAPI (blue) of SH-SY5Y cells transiently expressing myc-α-CD. Colocalization of DAPI and myc fluorescence is shown in pink (merge). (D) Immunocytochemistry of DIV 21 rat primary hippocampal cultures infected with rAAV expressing myc-α-CD together with Venus (myc-α-CD internal ribosome entry site Venus; Ven). Anti-Cα immunoreactivity is shown in red, direct Venus fluorescence in green, and DAPI staining in blue. The overlay of the three images is shown in the rightmost picture (merge). (E) Immunocytochemistry of DIV 21 rat primary hippocampal cultures infected with rAAV expressing Venus. Anti-Cα immunoreactivity is shown in red (absence of the red signal demonstrates the specificity of the Cα Ab), direct Venus fluorescence in green, and DAPI staining in blue. The overlay of the three images is shown in the rightmost picture (merge). Scale bars = 10 μm.