Ca2+ is essential for both synaptic vesicle exocytosis and endocytosis. In exocytosis it is
involved in controlling a number of different protein-protein interactions. In endocytosis one
Ca2+-dependent step is already identified, the Ca2+-calmodulin dependent phosphatase
calcineurin dephosphorylates a group of proteins called the dephosphins. The aim of this
thesis was to identify new Ca2+-dependent interactions that occur with endocytosis proteins
and to investigate their mechanism of action.
Endocytosis proteins contain a number of different specialised domains which control
protein-protein interactions at specific steps. A variety of these domains were used in a
proteomic screen using GST-pull down assays from nerve terminal lysates to investigate
whether Ca2+ controlled any specific interactions. Many interactions appeared to occur only
in the presence of Ca2+, however this was attributed to non-specific binding due to the
presence of ZnS04. Two new interactions were identified that bind SH3 domains only in the
absence of Ca2+, the adaptor protein caskin I and a protein known as "similar to KIAA0856".
Since SH3 domain containing proteins had Ca2+-dependent interactions, these were
examined in more detail. Most proteins that bound to SH3 domains had increased binding or
only bound in the absence of Ca2+. These proteins included caskin I, synaptojanin,
amphiphysin I, dynamin I and synapsin I. However an unidentified 90 kDa band bound only
in the presence of Ca2+ suggesting Ca2+ positively and negatively regulates interactions.
These interactions were characterised further by examining the amount of Ca2+ required to
stimulate or inhibit the above associations.
Ca2+-binding of endophilin II has been proposed to control its protein-protein interactions. A
number of complementary techniques were used to examine the Ca2+ affinity of SH3
domains including endophilin. These techniques included 45Ca2+ overlay assays, tyrosine
and tryptophan fluorescence and equilibrium dialysis. From all of the fusion proteins investigated the SH3 domains appeared to bind 45Ca2+ however the full length proteins did
not. Tyrosine and tryptophan fluorescence showed a Ca2+ dependent fluorescence shift
suggesting a conformational change on the addition of Ca2+, meaning that Ca2+ may bind to
the SH3 domains. Finally equilibrium dialysis showed that no endophilin GST fusion protein
bound to Ca2+. From these results it is still open to question whether endophilin binds Ca2+.