PTP-MEG2 regulates quantal size and fusion pore opening through two distinct structural bases and substrates (2021)
Figure 7

Figure 7. Dephosphorylations of MUNC18-1 at the pY145 site and DYNAMIN2 at the pY125 site by PTP-MEG2 determines the "foot" probability of catecholamine secretion from chromaffin cells.

(A). Structural representation and location of MUNC18-1-Y145 in the complex structure of MUNC18-1-SYNTAXIN1 (PDB: 3PUJ).

(B). Detailed structural representation of MUNC18-1-Y145 and its interactive residues. Y145 of MUNC18-1 interacts with the main chain carboxylic oxygen of residues I539 and G568 and forms hydrophobic interactions with L6, I539, L147, L179, L183, and L230 to tether the arc shape of the three domains of MUNC18-1 (PDB: 3PUJ).

(C). Association analysis of SNPs of MUNC18-1 with human disease.

(D). Interactions of the PTP-MEG2-trapping mutants with the MUNC18-1-Y145 mutants and DYNAMIN2-Y125 mutants. PC12 cells were transfected with FLAG-MUNC18-1-Y145, FLAG-DYNAMIN2-Y125 and different mutations of the FLAG-MUNC18-1-Y145A, Y145H, Y145E or Y145F, FLAG-DYNAMIN2-Y125A, Y125E, Y125F, 24 hours before stimulation with 100 nM AngII, respectively. The cell lysates were then incubated with GST-beads-PTP-MEG2-D470A complex for 2 hours with constant rotation. The potential PTP-MEG2 substrates were pulled down by GST-beads, and their levels were examined by the FLAG antibody with Western blot.

(E). Primary chromaffin cells were transduced with lentivirus containing the gene encoding wild-type MUNC18-1 or different mutants. These cells were stimulated with 100 nM AngII. The amperometric spikes were detected with electrochemical experiments. Typical amperometric traces are shown.

(F, G). The percentages of pre-spike foot (F) and stand-alone foot (G) for wild-type MUNC18-1 or different mutants were calculated.

(H). The MUNC18-1-Y145 mutations decreased the interaction between MUNC18-1 and SYNTAXIN1. PC12 cells were transfected with plasmid encoding SYNTAXIN-1. The proteins in cell lysates were pulled down with purified GST-beads-MUNC18-1-Y145 complex and the GST-MUNC18-1-Y145H/E/F, and detected with SYNTAXIN1 antibody. The right histogram shows the quantified protein levels.

(I). Primary chromaffin cells were transduced with lentivirus containing the gene encoding wild-type DYNAMIN2 or different mutants. These cells were stimulated with 100 nM AngII. The amperometric spikes were detected with electrochemical experiments. Typical amperometric traces are shown.

(J, K). The percentages of pre-spike foot (J) and stand-alone foot (K) for wild-type DYNAMIN2 or different mutants were calculated.

(L). Plasmids encoding DYNAMIN2 (DYNAMIN2-WT, DYNAMIN2-Y125E or DYNAMIN2-Y125F) and LYN-YFP, AT1aR-C-RLUC were co-transferred into HEK293 cells at 1:1:1 ratio. The BRET experiment was performed to monitor the mutation effects of DYNAMIN2 on AT1aR endocytosis in response to AngII (1μM) stimulation.

(M). The GTPase assay was performed to detect the GTPase activity of the DYNAMIN2-WT, DYNAMIN2-Y125E, DYNAMIN2-Y125F. Purified DYNAMIN2 proteins were incubated with 5μM GTP in the presence of 500μM DTT for 90 minutes, followed by addition of GTPase-Glo™ Reagent and detection reagent. The luminescence were measured after 37-minutes incubation.

(N). Phosphorylated Y125 of DYNAMIN2 was identified by LC-MS/MS. The PTP-MEG2-D470A trapping mutant was used to pull down potential PTP-MEG2 substrates from the adrenal lysates after AngII stimulation. Trypsin-digested potential PTP-MEG2 substrates were subjected to LC-MS/MS analysis. The doubly charged peptide with m/z 725.06 matches VYSPHVLNLTLIDLPGITK of DYNAMIN2 with Y125 phosphorylation.

Data information: (F-H, J-M) * indicates MUNC18-1, DYNAMIN2 mutants overexpression group compared with the WT overexpression group. # indicates MUNC18-1, DYNAMIN2 overexpression group compared with the control group. *, P<0.05; **, P<0.01; *** P<0.001 and #, P<0.05; ##, P<0.01; ### P<0.001. N.S. means no significant difference. Data were from 6 (F, G, J, K, L, M) or 3 (H) independent experiments. All the data were analyzed using one-way ANOVA, and showed as the mean ± s.e.m.