Proteolytic processing of protocadherin proteins requires endocytosis

Abstract

The α-, β-, and γ-protocadherins (Pcdhα, Pcdhβ, and Pcdhγ) comprise a large family of single-pass transmembrane proteins predominantly expressed in the nervous system. These proteins contain six cadherin-like extracellular domains, and proteolysis of Pcdhα and Pcdhγ by the γ-secretase complex releases their intracellular domains into the cytoplasm where they may function locally and/or enter the nucleus and affect gene expression. Thus, cleavage of Pcdhs may function to link intercellular contacts and intracellular signaling. Here we report that shedding of the Pcdhα extracellular domain and subsequent processing by γ-secretase require endocytosis and that Pcdhs interact with the regulator of vesicular sorting ESCRT-0 in undifferentiated cells. We also find that the accumulation of Pcdh cleavage products is regulated during development. Differentiation leads to an increase in the interactions between Pcdh proteins and a decrease in the accumulation of cleavage products. We conclude that Pcdh processing requires endocytosis and that the level of cleavage products is regulated during neuronal differentiation.

Fig. 1.

The levels of Pcdhα and -γ cleavage products decrease during both neuronal development and differentiation in culture. (A and B) Western blots of endogenous Pcdhα or Pcdhγ in whole brain extracts from mice at different developmental time points. Blots were probed with polyclonal antibody against the constant region of Pcdhα (αCon) or Pcdhγ (γCon). (C and D) Western blots of endogenous Pcdhα or Pcdhγ in cell lysates from primary neurospheres cultured for 4 d in vitro and treated with the γ-secretase inhibitor DAPT (+) or vehicle control (−). (E and F) Western blots of lysates from undifferentiated (UD) and differentiated (D) CAD cells stably expressing Pcdhα4-TAP or γb7-TAP. Cells were differentiated for 0 (UD) or 72 (D) h and the blots were probed with anti-FLAG and the loading controls anti-calnexin (CNX) or anti–β-actin. *Nonspecific, cross-reacting bands; CTF1, C-terminal fragment 1; FL, full-length Pcdh. An unidentified band in E that disappears during differentiation is indicated by an arrow.

Fig. 2.

Differentiation increases Pcdh–Pcdh binding. Coimmunoprecipitation of Pcdhα4-TAP and endogenous Pcdhβ and -γ is shown. Pcdhα4-TAP was immunoprecipitated from lysates of undifferentiated (UD) or differentiated (D) CAD cells and precipitates were probed with antibodies against FLAG, γCon, and the extracellular domain of Pcdhβ17.

Fig. 3.

Pcdh–Pcdh interactions play roles in cleavage suppression in differentiated CAD cells. (A) Schematic of full-length Pcdhα4-TAP (α4FL-TAP) and a mutant lacking the first five ECs (α4ΔEC1-5-TAP). (B) Coimmunoprecipitation of Pcdhα4-TAP fusions and endogenous Pcdhγ. α4FL-TAP or α4ΔEC1-5-TAP was precipitated from lysates of CADs differentiated for 24 h, and precipitates were blotted for γCon and FLAG. *IgG band in IP. (C) Western blot of cell lysates from undifferentiated and differentiated CAD cells stably expressing α4FL-TAP or α4ΔEC1-5-TAP.

Fig. 4.

Endocytosis is required for Pcdhα cleavage. (A) Western blot of cell lysates from CAD cells treated for 4 h with DMSO, lactacystin (Lac), or the cysteine protease inhibitor EST. (B) Western blot of cell lysates from CAD cells treated for 4 h with DMSO, the dynamin inhibitor dynasore (Dyna), or the weak base chloroquine (CQ). (C) Western blot of cell lysates from stably transfected CAD cells treated with lactacystin and/or dynasore for the indicated times.

Fig. 5.

Pcdhα is bound by ESCRT-0 in undifferentiated cells. (A) Confocal imaging of Pcdhα4-TAP and ESCRT-0. CAD cells stably expressing Pcdhα4-TAP were transfected with the ESCRT-0 subunit Hrs fused to GFP. After 0 or 72 h of differentiation, cells were fixed and stained with anti-HA (red or grayscale), anti-GFP (green or grayscale), and DAPI (blue). (B) Coimmunoprecipitation of Pcdhα4-TAP and endogenous Hrs. CAD cells stably transfected with an empty vector or Pcdhα4-TAP were differentiated for 0 h (UD) or 72 h (D). Pcdh was immunoprecipitated from cell lysates with anti-FLAG antibody, and Western blots were carried out for Hrs and FLAG. A nonspecific band comigrates with Pcdhα4-CTF1 following FLAG IP. (C) Coimmunoprecipitation of endogenous Hrs and Pcdhα4-TAP. Lysates of undifferentiated cells stably transfected with an empty vector or Pcdhα4-TAP were used for immunoprecipitation with a polyclonal antibody against Hrs or a control IgG. Western blots were then carried out against FLAG and Hrs. (D) Coimmunoprecipitation of α4ΔEC1-5-TAP and Hrs. α4ΔEC1-5-TAP was immunoprecipitated from lysates of undifferentiated or differentiated cells and a Western blot was performed against Hrs and FLAG.