Dynamin isoform-specific interaction with the shank/ProSAP scaffolding proteins of the postsynaptic density and actin cytoskeleton

Abstract

Dynamin is a GTPase involved in endocytosis and other aspects of membrane trafficking. A critical function in the presynaptic compartment attributed to the brain-specific dynamin isoform, dynamin-1, is in synaptic vesicle recycling. We report that dynamin-2 specifically interacts with members of the Shank/ProSAP family of postsynaptic density scaffolding proteins and present evidence that dynamin-2 is specifically associated with the postsynaptic density. These data are consistent with a role for this otherwise broadly distributed form of dynamin in glutamate receptor down-regulation and other aspects of postsynaptic membrane turnover.

Fig. 1. Dynamin isoform-specific association with Shank/ProSAP proteins and mapping of the binding sites via the yeast two-hybrid assay

a, chain of interactions involving Shank1 and dynamin. Interaction domains identified in previous studies or in this paper are indicated by bold arrows. SH, SH3 domain; GK, guanylate kinase domain; Ank, ankyrin repeats; CC, coiled-coil domain; PRD, proline-rich domain. b, yeast two-hybrid assay for dynamin interactions. Bait constructs were full-length wild-type dynamin-2 (Dyn-2), dynamin-1 (Dyn-1), and cytoplasmic dynein light intermediate chain 2 (LIC2). Prey constructs were the initially isolated 188-a.a. Shank1 fragment, full-length Shank2, and the JG4–5 vector (V). c, mapping of the Shank1, Shank2, and cortactin binding sites within dynamin-2. Carboxyl-terminal deletions of the dynamin-2 proline-rich domain were used as bait in yeast two-hybrid assays with Shank1, Shank2, and cortactin as prey. ++, very strong interaction; +, strong interaction; +/–, weak but detectable interaction; −, undetectable interaction. d, mapping of the dynamin-2 binding sites in Shank1. Full-length and amino-terminal deletions of the original 188-a.a. Shank1 prey fragment were assayed for interaction with the dynamin-2 bait in a yeast two-hybrid assay. e, sequence comparison between Shank1 and Shank2 using the Clustal W sequence alignment method of the putative dynamin-2 binding site. Within this 29-a.a. region, 72% of the amino acids are identical as highlighted.

Fig. 2. Co-immunoprecipitation of dynamin and Shank in COS7 cells

a, co-expression of full-length Shank2 with Myc-dynamin-1 or -2 in COS-7 cells. The dynamin isoforms were immunoprecipitated with anti-Myc, and the immunoprecipitates were immunoblotted with anti-Shank2 and anti-Myc. b, co-expression of Myc-tagged Shank1 with HA-tagged dynamin-1 or -2 in COS 7 cells. Shank1 was immunoprecipitated with anti-Myc, and the immunoprecipitates were blotted with anti-Myc and anti-HA antibodies. c, immunoprecipitation (i.p.) of dynamin-1 and -2 from rat brain cytosol using isoform-specific antibodies. The immunoprecipitates were blotted using anti-Shank2, anti-dynamin-1, and anti-dynamin-2 antibodies as indicated at right. d, anti-Myc immunoprecipitation of Myc-dynamin-1 and -2 singly expressed in COS-7 cells. The immunoprecipitates were detected using anti-Myc or anti-cortactin and showed no evidence for co-precipitation of cortactin with dynamin. B, beads alone control; D1, dynamin-1; D2, dynamin-2.

Fig. 3. Dynamin-2 interacts directly with the Shank/ProSAP family members

a, GST pull-down assays. Sepharose 4B beads, charged with either the GST-Shank2 fusion protein or GST alone, were incubated with purified recombinant HA-dynamin-2 and immunoblotted with an HA antibody. The upper panel shows that dynamin-2 directly interacts with GST-Shank2 (lane 2) but not with GST (lane 1), whereas the lower panel is the Coomassie-stained blot of the various purified GST proteins. −, absence of HA-dynamin-2; +, presence of HA-dynamin-2. b, blot overlay of Myc-tagged dynein intermediate chain 2C (DIC2C) or Myc-tagged Shank1 with purified recombinant HA-tagged dynamin-2. Shank1 and DIC2C were immunoprecipitated with an anti-Myc antibody and, after renaturation on the blot, overlaid with purified HA-dynamin-2 protein, which was detected with an anti-HA antibody. Upper panel, lanes 1 and 3, Myc-DIC2C and Myc-Shank1, respectively, in the absence of HA-dynamin-2; lanes 2 and 4, Myc-DIC2C and Myc-Shank1, respectively, overlaid with HA-dynamin-2. Lower panel, Coomassie-stained blots of the immunoprecipitated Myc-tagged proteins showing the location of each protein on the blot.

Fig. 4. Immunofluorescence microscopy of dynamin isoforms in hippocampal neurons

Rat hippocampal neurons (19 days in vitro) were double-labeled with anti-dynamin-1 or -2 antibodies versus anti-synaptophysin. Dynamin-2 exhibited punctate staining, which clearly overlapped with a subset of synaptophysin-positive spots (arrows). As a control, dynamin-1 also showed apparent co-localization with synaptophysin.

Fig. 5. Specific association of dynamin-2 with postsynaptic densities

Adult rat brain homogenate (H), synaptosomal (S), and postsynaptic density (P) fractions were immunoblotted and probed with anti-dynamin-1a, -1b, and -2, anti-synaptophysin (a presynaptic marker), and anti-Shank1, anti-calmodulin kinase II, anti-PSD-95, and anti-NMDAR1 (PSD markers).

Fig. 6. Co-expression of PSD proteins

When dynamin-2 was co-expressed with two components of the postsynaptic density, GKAP1a and Shank1a/ProSAP, it co-localized into clusters that were mediated by its interaction with Shank1a/ProSAP.