Developmental and Subcellular Profiles of Hippocalcin, AP2, and GluR2
(A) Hippocalcin expression in rat forebrain is developmentally regulated.
(B) Hippocalcin and AP2 have overlapping subcellular distributions in crude cell fractionations. GluR2 is enriched, and hippocalcin and AP2 are present in the synaptosomal (Syn) fraction.
(C-E) Presence of hippocalcin (n = 3), AP2 (n = 4), and GluR2 (n = 4) in progressively more stringent detergent extractions of the postsynaptic density in extraction buffer control (C), plus 2 mM EGTA (E), or plus 2 mM Ca²⁺ (Ca). Graphs show results of densitometry normalized to the PSDI Ca²⁺ condition.
(F) Presence of proteins in purified rat brain clathrin-coated vesicles (CCVs).
n ≥ 3 for all blots.

Schematic of the Role of Hippocalcin in Synaptically Evoked AMPAR Internalization
Ca²⁺ entry via NMDARs activates the hippocalcin myristyl switch and stimulates binding to β-adaptin of the AP2 complex, causing translocation of AP2-hippocalcin to the plasma membrane (A). AP2 binds to the GluR2 subunit of AMPARs and recruits clathrin (B). Clathrin displaces hippocalcin prior to endocytosis, after which AMPAR internalization occurs (C).

Hippocalcin Interacts with the N Terminus of the AP2 Subunit β2-Adaptin
(A) Hippocalcin is 193 residues long and contains an N-terminal myristoylation site and four EF-hand calcium-binding motifs. Full-length hippocalcin interacted with a clone consisting of the N-terminal region of the AP2 subunit β2-adaptin in the yeast two-hybrid. The N-terminal 72 amino acid residues of hippocalcin comprise the minimum interaction domain (HIP_2-72), interacting with full-length β2-adaptin.
(B) A177 and A172 anti-hippocalcin antibodies immunoprecipitate a single protein at the expected molecular weight for hippocalcin from rat forebrain as detected by the reciprocal anti-hippocalcin antibody in Western blots. Immunoprecipitation is prevented by the inclusion of the immunizing peptide or the replacement of antibody with preimmune serum.
(C) Anti-hippocalcin antibodies recognize a single specific band from rat forebrain extract.
(D) Ca²⁺ independence of TfR coimmunoprecipitation from rat forebrain with A177 anti-hippocalcin antibody. Due to the intensity of the band, the TfR input was detected separately. A177 immunization peptide reduced binding, demonstrating the specificity of the coimmunoprecipitation.
(E) Ca²⁺ dependence of AP2 and GluR2/3 coimmunoprecipitation from rat forebrain with A177 anti-hippocalcin antibody. The presence of both coprecipitating proteins is calcium dependent (2 mM added calcium) and is blocked by 2 mM EDTA. Inclusion of the immunization peptide or the presence of preimmune serum in place of the antibody also greatly diminished detection of β-adaptin or GluR2/3 in the immunoprecipitate.
(F) Inclusion of excess GST-HIP_2-72, but not GST alone, prevents detection of AP2 and GluR2 in A177 immunoprecipitates in the presence of 2 mM CaCl₂.
(G) In 14 days in vitro (DIV) hippocampal pyramidal neurons, hippocalcin immunoreactivity was detected throughout the cell, with some punctate localization in dendrites (see lower right panel). Scale bars, 20 μm and 5 μm in zoomed image.
(H) Double-label immunocytochemistry of 16 DIV cultured hippocampal neurons. Scale bars, 20 μm whole cell (left panel) and 5 μm dendritic region, respectively.
n ≥ 3 for all blots.

GST-HIP_2-72 Blocks the Induction of Hippocampal LTD but Has No Effect on Basal Transmission or LTP
Pooled data for whole-cell patch-clamp recordings from CA1 pyramidal cells in hippocampal slices. Infusion of GST ([A]; n = 12) or GST-HIP_2-72 ([B]; n = 24) has no effect on EPSC amplitude during basal stimulation. Homosynaptic LTD is induced in cells infused with GST ([C], n = 8); however, LTD is blocked in cells infused with GST-HIP_2-72 ([D]; n = 9). LTP is induced in cells infused with GST ([E], n = 8), and a similar amount of LTP is induced in cells infused with GST-HIP_2-72 ([F]; n = 6). For (C)-(F), the insets (top) show EPSCs collected at the times indicated from example experiments. Plasticity induction protocol applied to pathway represented by filled symbols at the time indicated by the black bar.