Interactions Regulating Cell Signaling.
soluble proteins or enzymes involved in signal transduction become
attached to membrane interfaces as a result of protein acylation
and or electrostatic interactions with negatively charged lipid
interfaces. For example, MARCKS (the myristolated alanine-rich
C-kinase substrate), requires both acylation and electrostatic
interactions to become associated with the plasma membrane, it
appears to regulate phosphoinositides (such as PI(4,5)P2) within
also become attached as a result of the interaction of protein
domains, such as C2 domains (4) or PH domains (5) with the membrane
interface. C2 domains bind membranes in a Ca2+-dependent fashion
and appear to be one of the most abundant Ca2+-regulatory motifs
found in eucaryotic systems. PH (plexstrin homology) domains
bind to polyphosphoinosites and show specificity for specific
phosphorylated inositol headgroups.
attachment and defects in membrane binding domains are involved
in many disease states. Dysferlin is a protein that contains
6 C2 domains and it has been implicated in membrane repair in
muscle. A deficiency in this protein or mutations in one of its
C2 domains appear to be responsible for certain forms of muscular
dystrophy (6,7). Otoferlin also has 6 C2 domains and is likely
also involved in membrane repair. A mutation in the third C2
domain of human otoferlin is believed to alter its Ca2+ binding
ability and has been linked to an inherited form of deafness
(8). Many oncogenes express aberrant proteins involved in cell-signaling,
and in some cases membrane binding has been shown to be of central
importance. For example, the viral form of the src tyrosine kinase
will transform cells and promote cancer, but variants of the
protein that fail to bind membranes do not transform cells (9).
An Arg to Gln mutation in the PX domain of p47phox prevents targeting
of the NADPH oxidase to membranes containing PI(3)P. This is
responsible for chronic granulomatous, an immunodeficiency disease
associated with reduced superoxide production (10).
work is addressing the following questions regarding the function
of these domains:
are membrane binding domains oriented on the membrane interface,
how deeply do they penetrate into the bilayer, and what are
the forces involved in attachment?
are the mechanisms for Ca+2-induced translocation of C2 domains
to the membrane interface. How do electrostatic, hydrophobic
and entropic factors contribute to the binding?
are the membrane bound structures for C2 domains. Does Ca2+-binding
alter the structure of the domain or create a binding site
do the C2 domains from synaptotagmin dimerize, interact with
components of the SNARE complex, and how do they regulate neuronal
have been addressing these questions with site-directed spin
labeling and high-resolution NMR. For example, we recently determined
the orientation and depth of penetration of C2 domains from cPLA2
and synaptotagmin at the membrane interface (11-13). The interactions
made by the first synaptotagmin domain (C2A) is in contrast to
that made by the C2 domain from cPLA2, which has a similar orientation
but is translocated about 5Å deeper into the membrane hydrocarbon.
This difference in depth is consistent with the results of computational
studies, which predict that long-range electrostatic interactions
and desolvation will make different contributions to the membrane
binding of these domains.
current work on C2 domains includes measurements on the second
C2 domain from synaptotagmin (C2B) and measurements on larger
fragments of synaptotagmin including both C2 domains and the
transmembrane anchor. Synaptotagmin appears to be the primary
protein mediating the Ca2+-triggered exocytosis in neurons. We
are now setting up experiments to map protein-protein interactions
between synaptotagmin and proteins that make up the SNARE complex
and to determine how C2 domains might modulate bilayer curvature
strain. Some of these experiments will make use of EPR distance
measurements between labeled sites in the interacting proteins,
and will cover medium and longer range distances (7 to 40Å).
addition to these EPR based experiments, we are making novel
NMR measurements on the electrostatic surface potential of the
synaptotagmin C2A domain by examining paramagnetic enhancements
of nuclear relaxation produced by charged nitroxides or metal
ion complexes. These measurements will be compared with the predictions
of computational approaches. We are also exploring the use of
NMR and bicelle systems to recover residual dipolar couplings
for surface associated C2 domains. These data will be used to
provide information on the orientation of the membrane associated
domain as well as its membrane bound structure.
- Rauch, M.E., C.G. Ferguson, G.D. Prestwich, and D.S. Cafiso,
Myristoylated alanine-rich C kinase substrate (MARCKS) sequesters
spin-labeled phosphatidylinositol 4,5-bisphosphate in lipid
bilayers. J Biol Chem, 2002. 277(16): p. 14068-76.
- Wang, J., A. Gambhir,
G. Hangyas-Mihalyne, D. Murray, U. Golebiewska, and S. McLaughlin,
Lateral sequestration of phosphatidylinositol 4,5-bisphosphate
by the basic effector domain of myristoylated alanine-rich C kinase substrate
is due to nonspecific electrostatic interactions. J Biol Chem, 2002.
277(37): p. 34401-12.
- Gambhir, A., G. Hangyas-Mihalyne, I. Zaitseva, D.S. Cafiso,
J. Wang, D. Murray, S.N. Pentyala, S.O. Smith, and S. McLaughlin,
of PIP2 on phospholipid membranes by basic/aromatic regions of proteins.
Biophys J, 2004. 86(4): p. 2188-207.
- Rizo, J. and T.C. Sudhof, C2-domains,
structure and function of a universal Ca2+-binding domain.
J. Biol. Chem., 1998. 273: p. 15879-15882.
- Lemmon, M.A. and K.M.
Ferguson, PH domains: diverse sequences with a common fold
recruit signaling molecules to the cell surface.
85: p. 621-624.
- Davis, D.B., K.R. Doherty, A.J. Delmonte, and E.M.
McNally, Calcium-sensitive phospholipid binding properties
of normal and mutant ferlin C2 domains.
J Biol Chem, 2002. 277(25): p. 22883-8.
- Bansal, D. and K.P. Campbell,
Dysferlin and the plasma membrane repair in muscular dystrophy.
Trends Cell Biol, 2004. 14(4): p.
- Mirghomizadeh, F., M. Pfister, F. Apaydin, C. Petit,
S. Kupka, C.M. Pusch, H.P. Zenner, and N. Blin, Substitutions
in the conserved
domain of otoferlin
cause DFNB9, a form of nonsyndromic autosomal recessive deafness.
Neurobiol Dis, 2002. 10(2): p. 157-64.
- Sigal, C.T., W. Zhou,
C.A. Buser, and S.A. McLaughlin, Amino-terminal basic residues
of Src mediate membrane binding through electrostatic
interaction with acidic phospholipids. PNAS, 1994. 91:
- Noack, D., J. Rae, A.R. Cross, B.A. Ellis, P.E. Newburger,
J.T. Curnutte, and P.G. Heyworth, Autosomal recessive
chronic granulomatous disease caused
by defects in NCF-1, the gene encoding the phagocyte
p47-phox: mutations not arising in the NCF-1 pseudogenes. Blood,
2001. 97(1): p. 305-11.
- Frazier, A.A., M.A. Wisner, N.J. Malmberg,
K.G. Victor, G.E. Fanucci, E.A. Nalefski, J.J. Falke, and
Membrane orientation and
the C2 domain from cPLA2 by site-directed spin labeling.
Biochemistry, 2002. 41: p. 6282-6292.
- Frazier, A.A.,
C.R. Roller, J.J. Havelka, A. Hinderliter, and D.S. Cafiso,
Membrane-bound orientation and position
of the synaptotagmin
I C2A domain by
site-directed spin labeling. Biochemistry, 2003.
42(1): p. 96-105.
- Rufener, E., A. Frazier, C.M. Wieser,
A. Hinderliter, and D.S. Cafiso, Membrane bound orientation
synaptotagmin C2B domain
determined by site-directed spin labeling. Biochemistry,
2005. 44: p. 18-28.