Changes in Biological Activity and Folding of Guanylate Cyclase-Activating
Protein 1 as a Function of Calcium
†
Maria Rudnicka-Nawrot,
‡
Irina Surgucheva,
§
Jeffrey D. Hulmes,
|
Franc ¸ oise Haeseleer,
‡
Izabela Sokal,
‡
John W. Crabb,
|
Wolfgang Baehr,
§
and Krzysztof Palczewski*
,‡,⊥
Departments of Ophthalmology and Pharmacology, School of Medicine, UniVersity of Washington,
Seattle, Washington 98195, Moran Eye Center, UniVersity of Utah Health Science Center, Salt Lake City, Utah 84132,
and W. Alton Jones Cell Science Center, Lake Placid, New York 12946
ReceiVed September 16, 1997; ReVised Manuscript ReceiVed October 23, 1997
X
ABSTRACT: Guanylate cyclase-activating protein 1 (GCAP1), a photoreceptor-specific Ca
2+
-binding protein,
activates retinal guanylate cyclase 1 (GC1) during the recovery phase of phototransduction. In contrast
to other Ca
2+
-binding proteins from the calmodulin superfamily, the Ca
2+
-free form of GCAP1 stimulates
the effector enzyme. In this study, we analyzed the Ca
2+
-dependent changes in GCAP1 structure by
limited proteolysis and mutagenesis in order to understand the mechanism of Ca
2+
-sensitive modulation
of GC1 activity. The change from a Ca
2+
-bound to a Ca
2+
-free form of GCAP1 increased susceptibility
of Ca
2+
-free GCAP1 to proteolysis by trypsin. Sequencing data revealed that in the Ca
2+
-bound form,
only the N-terminus (myristoylated Gly
2
-Lys
9
) and C-terminus (171-205 fragment) of GCAP1 are
removed by trypsin, while in the Ca
2+
-free form, GCAP1 is readily degraded to small fragments. Successive
inactivation of each of the functional EF loops by site-directed mutagenesis showed that only EF3 and
EF4 contribute to a Ca
2+
-dependent inactivation of GCAP1. GCAP1(E
75
D,E
111
D,E
155
D) mutant did not
bind Ca
2+
and stimulated GC1 in a [Ca
2+
]-independent manner. GCAP1 and GCAP2, but not S-100,
a high [Ca
2+
]
free
activator of GC1, competed with the triple mutant at high [Ca
2+
]
free
, inhibiting GC1 with
similar IC
50
’s. These competition results are consistent with comparable affinities between GC1 and
GCAPs. Our data suggest that GCAP1 undergoes major conformational changes during Ca
2+
binding
and that EF3 and EF4 motifs are responsible for changes in the GCAP1 structure that converts this protein
from the activator to the inhibitor of GC1.
Calcium ions, Ca
2+
, play a crucial role in cellular signaling.
Because they are nondegradable, several systems have
evolved to regulate the cellular concentration of free Ca
2+
([Ca
2+
]
free
), including intracellular compartmentalization/
sequestration, pumping to the extracellular space, and buffer-
ing by Ca
2+
-binding proteins. Some of these Ca
2+
-binding
proteins are also poised to take advantage of transient
changes in [Ca
2+
]
free
to affect properties of regulatory
enzymes and ion channels. In cells that lower their internal
[Ca
2+
]
free
upon excitation, such as rod and cone photoreceptor
cells, distinct types of proteins have evolved that act as
activators of effector enzymes when they are in the Ca
2+
-
free state. Guanylate cyclase-activating proteins, GCAP1
1
and GCAP2, were found to fulfill such functions in the
regulation of photoreceptor guanylate cyclase (GC1) (1-
4).
GCAP1 and GCAP2 are acidic, ∼23-kDa, homologous
proteins that contain three functional high-affinity, EF-hand
Ca
2+
-chelating motifs (reviewed in ref 5). At low [Ca
2+
]
free
,
GCAPs increase the activity of GC1 (6) at least 10-fold (1)
by an unknown mechanism. GCAP1 forms a stable complex
with GC1, independent of [Ca
2+
]
free
. The GC1/GCAP1
complex may switch between two conformations, active and
inactive, with the binding or dissociation of Ca
2+
(2, 7).
GCAP2 may translocate from the cytosol to the membrane-
bound cyclase when it is free of Ca
2+
and stimulates GC1
activity (8). The N-terminal fatty acid-acylated regions of
both GCAPs show weak sequence conservation, and the
function of this region remains speculative. It is possible
that the N-terminus is flexible and exposed, providing
hydrophobic tethering to the membranes for the most
efficient stimulation of GC1, as proposed for GCAP1 by
Otto-Bruc et al. (9), but this modification is functionally
unrelated in the GC1 stimulation by GCAP2, as proposed
by Olshevskaya et al. (8).
†
This research was supported by grants from NIH EY08061,
EY06603, EY08123; Core Facilities Grants EY01730 and an award
from Research to Prevent Blindness, Inc. (RPB), to the Department of
Ophthalmology at the University of Washington, and the University
of Utah. KP is a recipient of a Jules and Doris Stein Professorship
from RPB.
* Address correspondence to this author at the University of
Washington, Department of Ophthalmology, Box 356485, Seattle, WA
98195-6485. Phone: 206-543-9074. Fax: 206-543-4414.
‡
Department of Ophthalmology.
§
University of Utah Health Science Center.
|
W. Alton Jones Cell Science Center.
⊥
Department of Pharmacology.
X
Abstract published in AdVance ACS Abstracts, December 1, 1997.
1
Abbreviations: BTP, 1,3-bis[[tris(hydroxymethyl)methyl]amino]-
propane; GC1, photoreceptor guanylate cyclase 1; GCAP, guanylate
cyclase-activating protein; ROS, rod outer segments.
248 Biochemistry 1998, 37, 248-257
S0006-2960(97)02306-4 CCC: $15.00 © 1998 American Chemical Society
Published on Web 01/06/1998