99

Pharmacological Research, Vol. 25, Supplement 1, 1992

SIGNAL TRANSDUCTION AT THE NERVE TERMINAL LEVEL : ROLE OF THE PHOSPHORYLATION OF SYNAPTIC VESICLE PROTEINS F . Valtorta§ and F . Benfenati' s"B . Ceccarelli Center", CNR Center of Cytopharmacology, Dept . of Medical Pharmacology, Univ . of Milan ; `Dept . of Human Physiology, Univ . of Modena Introduction

The

most widely accepted hypothesis

to

explain the quantal

each quantum is confined within one synaptic vesicle and is released by exocytosis when the vesicle nature

of

neurotransmitter release

membrane fuses with the axolemma .

holds that

Quantal release is a dynamic phenomenon,

since it is influenced at any given moment by previous activity,

i .e . the

number

previous

of

quanta

frequency and modulability

released following

pattern of stimulation .

a

stimulus

depends

on

the

One possible explanation

of neurotransmitter release upon activity

is

for the

that

this

modulation is brought about by variations in the intra-terminal levels of second messengers (1) . Second messengers are thought to modulate neurotransmitter release by activating specific enzymes, the protein kinases, which in turn phosphorylate specific protein substrates .

Among the phosphoproteins

present in nerve terminals, a key role in the process of neurotransmitter release is probably played by those which are located on the membrane of synaptic vesicles . Therefore, the study of protein kinases and phosphoproteins associated with synaptic vesicles may allow the elucidation of the processes that lead to the fusion of the synaptic vesicle membrane with the axolemma in response to membrane depolarization and of the processes that modulate the release of neurotransmitter during repetitive stimulation . Moreover, synaptic vesicle-specific proteins can be used as selective markers for the synaptic vesicle membrane in immunocytochemical studies . Four serine phosphoproteins collectively called synapsins (synapsin Ia, Ib, IIa and IIb) represent a family of differentially spliced synaptic vesicle-associated proteins . The synapsins are major substrates for cAMPdependent protein kinase and Ca"/ calmodulin-dependent protein kinase I . In addition, synapsins Ia and Ib possess a highly elongated and basic tail which contains two additional phosphorylation sites for Ca 2+/ calmodulindependent protein kinase II . Synapsin I binds to the cytoplasmic side of the synaptic vesicle membrane and to F-actin and both interactions are weakened by phosphorylation of synapsin I by Ca t '/calmodulin-dependent protein kinase II (4) . Materials and Methods Synapsin I was purified and phosphorylated as described by Schiebler et al . (JBC 261 :8383-8390) and modified by Bahler and Greengard (Nature 326 :704-707) . Actin was purified from rabbit skeletal muscle as described by Spudich and Watt (JBC 246 :4866-4871) . Synaptic vesicles were purified from rat forebrain as described by Huttner et al . (JCB 96 :1374-1388) . Synapsin I fragments were prepared and purified according to Bahler et al . (JCB 108 :1841-1849) . The binding of synapsin I and its fragments to actin and to synaptic vesicles was tested by ultracentrifugation . Actin bundling was analyzed by electron microscopy and

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© 1992 The Italian Pharmacological Society

Pharmacological Research, Vol. 25, Supplement 1, 1992

100

light scattering . The actin-nucleating activity was determined by following the kinetics of actin polymerization using pyrenyl-actin and by measuring the binding of cytochalasin B to actin filaments . Results

we

have

purified

cysteine-specific cleavage structure-function

and

analysis .

synapsin I molecule

which

fragments have

We

of synapsin I

used

have identified

bind

to

generated

these fragments

actin

and

to

distinct

to

the

by

perform

sites

in

synaptic

a

the

vesicle

membrane, respectively . These results are compatible with the possibility of forming a ternary complex synapsin I-synaptic vesicle-actin . We

have

also

tested the

formation

of

the

ternary

complex

by

measuring the effects determined by synapsin I on the state of polymerization / aggregation of actin . We have found that synapsin I has a nucleating effect synapsin I

on actin .

The

effect

on

actin

is

still

is bound to the synaptic vesicle membrane,

present when

indicating that

it

can interact contemporarily with both structures . The ability of synapsin I to interact with actin and with synaptic vesicles is regulated by site-specific phosphorylation TableI ACTIN BINDING

ACTIN BUNDLING

(Table I) .

ACTIN NUCLEATION

VESICLE BINDING

DEPHOSPHOSYNAPSIN I

++++

++++

++++

++++

PHOSPHOSYNAPSIN I (site 1)

+++

+++

++

++++

PHOSPHOSYNAPSIN I (sites 2,3)

++

Discussion

These

I plays a

synapsin

results

are

role

the

in

consistent

with

regulation

the hypothesis

of neurotransmitter

that

release,

by reversibly cross-linking synaptic vesicles to the actin filaments within the nerve terminal . According to this hypothesis, under resting conditions, when most of the synapsin I molecules are possibly

dephosphorylated, synapsin I fusion,

by

stimulation, dependent

protein

phosphorylation of ternary from

the

represents

an

inhibitory

constraint

complex

kinase

II, leading

to

an

increase

in

the

state

of

synapsin I .

synapsin

This would cause the dissociation of the I-synaptic vesicle-actin, releasing the vesicle

cytoskeleton and

allowing

it

to

become

part

synaptic vesicles which are available for fusion . References 1)

for

tethering synaptic vesicles to the cytoskeleton . Upon Cat, flows into the nerve terminal activating Ca"/ calmodulin-

F . Valtorta, R . Fesce, F . Grohovaz, C . Haimann, W .P . Hurlbut, N . Iezzi, F . Torri-Tarelli, A . Villa and B . Ceccarelli . Neuroscience, 35 : 477-489, 1990,

2) P . De Camilli, F . Benfenati, F . Valtorta and P . Greengard . Annual Review of Cell Biology, 6 : 433-460, 1990 .

of

the pool

of