Sharma2and Karl-Wilhelm Koch1 1 Institut fu¨r Biologische Informationsverarbeitung 1, Forschungszentrum Ju¨lich, Ju¨lich, Germany;2The Unit of Regulatory and Molecular Biology, Departmen
Trang 1Regulatory modes of rod outer segment membrane guanylate cyclase differ in catalytic efficiency and Ca2+-sensitivity
Ji-Young Hwang1,*, Christian Lange1,†, Andreas Helten1, Doris Ho¨ppner-Heitmann1, Teresa Duda2,
Rameshwar K Sharma2and Karl-Wilhelm Koch1
1
Institut fu¨r Biologische Informationsverarbeitung 1, Forschungszentrum Ju¨lich, Ju¨lich, Germany;2The Unit of Regulatory and Molecular Biology, Departments of Cell Biology andOphthalmology, NJMS & SOM, UMDNJ, Stratford, NJ, USA
In rod phototransduction, cyclic GMP synthesis by
mem-brane bound guanylate cyclase ROS-GC1 is under Ca2+
-dependent negative feedback control mediated by guanylate
cyclase-activating proteins, GCAP-1 and GCAP-2 The
cellular concentration of GCAP-1 and GCAP-2
approxi-mately sums to the cellular concentration of a functional
ROS-GC1 dimer Both GCAPs increase the catalytic
effi-ciency (kcat/Km) of ROS-GC1 However, the presence of a
myristoyl group in GCAP-1 has a strong impact on the
regulation of ROS-GC1, this is in contrast to GCAP-2
Catalytic efficiency of ROS-GC1 increases 25-fold when it is
reconstituted with myristoylated GCAP-1, but only by a
factor of 3.4 with nonmyristoylated GCAP-1 In contrast to
GCAP1, myristoylation of GCAP-2 has only a minor effect
on kcat/Km The increase with both myristoylated and non-myristoylated GCAP-2 is 10 to 13-fold GCAPs also confer different Ca2+-sensitivities to ROS-GC1 Activation of the cyclase by GCAP-1 is half-maximal at 707 nMfree [Ca2+], while that by GCAP-2 is at 100 nM The findings show that differences in catalytic efficiency and Ca2+-sensitivity of ROS-GC1 are conferred by GCAP-1 and GCAP-2 The results further indicate the concerted operation of two
GCAP modes that would extend the dynamic range of cyclase regulation within the physiological range of free cytoplasmic Ca2+in photoreceptor cells
Keywords: phototransduction; guanylate cyclase; GCAP; myristoylation; kcat/Km
Photoexcitation of vertebrate photoreceptor cells leads to
the hydrolysis of cyclic GMP (cGMP) and subsequent
closure of the cyclic nucleotide-gated (CNG) channels in the
plasma membrane Restoration of the dark state of the
photoreceptor cell requires the reopening of CNG-channels
(reviewed in [1–3]) A critical step in this recovery process is
synthesis of the second messenger, cGMP Studies with
vertebrate photoreceptor cells, constituting mainly rods,
show that these cells express two types of a membrane
bound guanylate cyclase termed ROS-GC1 and ROS-GC2
(alternatively used names are retGC1 and retGC2 and
GC-E and GC-F; reviewed in [4,5]) ROS-GC1 has been
purified directly from bovine and amphibian rod outer
segments [6–9], and it is the only cyclase which has been cloned based on its amino acid sequence [8,10] Human retinal diseases (LCA1 and CORD6) affect both rod and cone vision, but are only linked to the ROS-GC1 gene [11–17] Knowledge about enzyme kinetic parameters of native photoreceptor guanylate cyclase are so far restricted
to ROS-GC1 This is mainly because only ROS-GC1 has been purified from bovine retina and thus, probably, constitutes the main cyclase in bovine rod outer segment preparations Reported Km-values for the substrate, GTP, range from 0.76–1.1 mM[6,7,9,18] Turnover numbers (kcat)
of the purified enzyme range from 0.2–3.9 cGMPÆs)1[6,9] Small acidic Ca2+-binding proteins, called guanylate cyclase-activating proteins or GCAPs, regulate ROS-GC1 Three GCAP (GCAP-1, 2 and 3) isoforms have been cloned from retinal sources [19–23] GCAP-1 and GCAP-2 are both expressed in rod and cone cells of different species as shown by immunocytochemistry [21,22,24,25] Expression
of GCAP-3 is more restricted; it is present in human cones, fish rods and cones, but not in mice photoreceptor cells [26] Thus, GCAP-3 does not appear to be a general sensor of
Ca2+-pulses linked with phototransduction
GCAP-1 and GCAP-2 contain one nonfunctional and three functional EF-hands Through functional hands they detect changes in the intracellular Ca2+-concentration [Ca2+] and modulate ROS-GC1 Dark adapted vertebrate photoreceptor cells have a cytoplasmic free [Ca2+] of 500–
650 nM This falls below 100 nMupon illumination [27–30] GCAPs detect the fall and in their Ca2+-free form, activate ROS-GC1 [4,5,19–23] The generated cyclic GMP replen-ishes the depleted pool and restores the channels in their open state While there is wide agreement in the literature
Correspondence to K-W Koch, Institut fu¨r Biologische
Information-sverarbeitung 1, Leo-Brandt-Strasse, Forschungszentrum Ju¨lich,
D-52425 Ju¨lich, Germany.
Fax: + 49 2461 614216, Tel.: + 49 2461 61-3255,
E-mail: k.w.koch@fz-juelich.de
Abbreviations: ROS, rod outer segments; ROS-GC1/GC2,
photo-receptor membrane guanylate cyclases 1 or 2; GCAP-1/2, guanylate
cyclase activating protein 1 or 2; NMT, N-terminal myristoyl
trans-ferase; myr, myristoylated; nonmyr, nonmyristoylated; Rh, rhodopsin.
Enzymes: guanylate cyclase (EC 4.6.1.2.)
*Present address: Genetics & Molecular Biology Branch National
Human Genome Research Institute National Institute of Health Bldg.
49, Rm 4A08, 49 Convent Drive, Bethesda, MD 20892–4442, USA.
Present address: Instituto de Bioquı´mica Vegetal y Fotosı´ntesis,
Centro de Investigaciones Isla de la Cartuja, Avda Ame´rico Vespucio
s/n, 41092 Sevilla, Spain.
(Received 2 June 2003, accepted 28 July 2003)
Trang 2that these Ca2+-binding-proteins are powerful activators of
the ROS-GCs [4,5,19–23], there is no unanimity on their
specific expression in rods or cones and to which ROS-GC
they are paired with Some immunocytochemical studies
show that GCAP-1 is the predominant form in cones and
GCAP-2 in rods [31,32]; and recently the physiological role
of GCAP-2 has been questioned, because the expression of
GCAP-2 in transgenic GCAPs null mice did not fully
restore the normal flash response of the wild-type [33]; and
Howes et al [34] in a subsequent study on transgenic mice
showed, that the wild-type phenotype flash response could
be rescued by the expression of GCAP-1 in the absence of
GCAP-2
To better define the Ca2+-modulated ROS-GC
trans-duction component in rod cells we addressed the following
questions in the present work: how much of GCAP-1 and
GCAP-2 is present in rod outer segments? Are there
differences between GCAP-1 and GCAP-2 in their
regula-tory properties and if so, what is the physiological
signifi-cance of such a difference?
The study demonstrates that GCAP1 and GCAP2
modulate the Ca2+signaling of ROS-GC1 in different ways
Experimental procedures
Preparation of ROS
Bovine ROS were prepared according to a standard
protocol under dim-red light This involved sucrose density
centrifugation in the presence of a moderate salt
concen-tration to minimize loss of cytoplasmic proteins [35] ROS
were at all time stored and handled in the dark Rhodopsin
(Rh) concentration was determined
spectrophotometri-cally at 498 nm using a molar extinction coefficient of
40 000M )1Æcm)1
Expression and purification of GCAPs
GCAP-1 and GCAP-2 were expressed in E coli and
purified as described previously [36,37] Myristoylated
(Myr) forms of GCAP-1 and GCAP-2 were obtained by
their coexpression with yeast N-myristoyl-transferase
(NMT) (the plasmid pBB131 was kindly provided by
J Gordon, Washington University School of Medicine, St
Louis, MO, USA) As wild-type GCAP-1 does not contain
a consensus site for yeast NMT, we used the mutant D6
S-GCAP-1 that is functionally indistinguishable from myr
GCAP-1 [38] The degree of myristoylation was determined
by HPLC
Heterologous expression of ROS-GC1
HEK293 tsA cells HEK293 tsA cells were grown and
transfected with ROS-GC1 in pcDNA3.1 expression vector
by the calcium phosphate method [16] The following
modifications were applied: 22 h post-transfection cells were
washed sequentially with NaCl/Pi, NaCl/Piplus EDTA and
NaCl/Piand incubated in medium for 20–22 h Cells were
harvested by a short centrifugation step (200 g; 5 min;
4C), resuspended in NaCl/Piand centrifuged again The
pellet was resuspended in lysis buffer (10 mMHepes/KOH
pH 7.5, 1 m dithiothreitol), sonicated and centrifuged to
remove cellular debris (400 g; 5 min; 4C) The supernatant was then centrifuged at 125 000 g for 15 min at 4C to pellet the membranes The membranes were resuspended in
10 mM Hepes/KOH pH 7.5, 250 mM KCl, 10 mM NaCl and 1 mMdithiothreitol Protein concentration was deter-mined by the amido black method [39]
COS7 cells COS7 cells were transfected with the wild-type recombinant ROS-GC1 cDNA in pcDNA3 expression vector by the calcium phosphate coprecipitation technique
as described before [16] Sixty hours after transfection, cells were washed twice with 50 mM Tris/HCl pH 7.5 buffer They were then scraped into 2 mL cold buffer, homogen-ized, centrifuged for 15 min at 5000 g and washed sev eral times with the same buffer
Electrophoresis and Western blotting SDS/PAGE and Western blotting were performed accord-ing to previously defined protocols [16,22]
Chemiluminescence detection and quantitation
of proteins Quantitative chemiluminescence analysis was performed
on a Luminograph LB 980 (Berthold) Varying amounts (10–50 ng) of ROS-GC1 were electrophoresed with a sample
of ROS or ROS membranes After SDS/PAGE, proteins were blotted and probed with anti-ROS-GC1 Igs Shortly before measurement, the blots were incubated with Western blotting ECL reagent 1 and 2 (Amersham) for 1 min and photon emission per second was detected at 480 nm Varying amounts of ROS-GC1 were used for calibration and the amount of cyclase in ROS was obtained directly from the calibration curve The predominant membrane guanylate cyclase in bovine ROS is ROS-GC1, the other isoform (ROS-GC2) that is expressed in photoreceptor cells consti-tutes less than 10% of the amount of ROS-GC1 (A Helten and K.-W Koch, unpublished observation) Amounts of GCAPs were obtained by a similar proce-dure but instead of a Luminograph we used a Kodak Image Station Exact amounts of purified GCAP-1 or GCAP-2 for SDS/PAGE and Western blotting were determined from GCAP specific protein standard curves These curves were created as follows: purified preparations of GCAP-1 and GCAP-2 were used to determine their molar extinction coefficients as described in [40] The values were
e280¼ 28378M )1Æcm)1 for GCAP-1 and e280¼ 37512
M )1Æcm)1for GCAP-2 Exact stock solutions of GCAP-1 and GCAP-2 were prepared using the molar absorbance coefficients and a calibration curve was made using the Coomassie Blue dye binding assay GCAP solutions were adjusted every time by the use of these GCAP-1 or GCAP-2 specific calibration curves
Guanylate cyclase assay For reconstitution experiments, pure ROS were washed several times in low salt buffer and the membranes were prepared as described in [22] GCAPs were reconstituted with washed ROS membranes and guanylate cyclase activity was determined as described in [22] Determination
Trang 3of guanylate cyclase activities in HEK and COS cell
membranes was as described previously [16] Incubation
of ROS-GCs at a constant concentration of 2 mMEGTA
and varying concentration of GCAPs (0–10 lM) were
performed to obtain EC50values for the GCAP Keeping
the GCAP concentration constant while varying free [Ca2+]
yielded IC50values of Ca2+for each GCAP
Analysis of enzyme kinetics
ROS-GC1 in washed ROS membranes was reconstituted
with 2 lMof purified myr- or nonmyr-forms of GCAP-1
and GCAP-2 Guanylate cyclase activity was assayed at
2 mM EGTA (£ 10 nM free [Ca2+]) as a function of the
substrate GTP We used Mg2+as cofactor and kept the
ratio of Mg2+ to GTP at 5 : 1 Analysis of data was
performed withORIGIN6.1 andSIGMA PLOT4.2 software A
direct plot of activity vs [GTP] gave in all cases a sigmoidal
curve indicating cooperative substrate binding In order to
analyze data of a sigmoidal dependence in a linear
Lineweaver–Burk plot [41], we determined a Hill coefficient
nfrom a fit of the direct plot Values of Vmaxand Kmwere
then determined from a plot of 1/V (reciprocal of guanylate
cyclase activity) vs 1/[S]n (reciprocal of the substrate
concentration raised to the power of n)
Preparation of Ca2+-buffer and determination of free
Ca2+concentration
Free [Ca2+] concentrations of buffer solutions were
calcu-lated using the programWEBMAX2.0 (Stanford University,
CA, USA) The free [Ca2+] of guanylate cyclase assay
buffer solutions was checked at 30C with a Ca2+-sensitive
electrode (World Precision Instruments, Inc.) using
calibra-tion standards in the range from 10)8to 10)1Mfree [Ca2+]
Results
The Ca2+-sensor proteins GCAP-1 and GCAP-2
are almost equally expressed in native bovine ROS
membranes
Molar concentrations of GCAPs have not been reported so
far, although they are indispensable for a full quantitative
description of phototransduction We determined these
values by the following procedure: 0.5–9 ng of purified
GCAP-1 and 2–10 ng of GCAP-2 were loaded onto PAGE
ROS containing 5–25 lg of rhodopsin were loaded on the
same gel After electrophoresis, proteins were
electrotrans-ferred to a blot membrane and probed with antibodies
against GCAP-1 or GCAP-2 Bands were visualized by
chemiluminescence Chemiluminescence intensity was
line-arly dependent on the amount of antigen and was used to
quantify the amounts of GCAPs An example is shown in
Fig 1A,B, where the amount of GCAP standards showed a
linear increase in chemiluminescence intensity The analysis
of several Western blots revealed a ratio of 1 : 1200 ± 360
(N¼ 12) of GCAP-1 to rhodopsin and 1 : 1100 ± 560
(N¼ 5) of GCAP-2 to rhodopsin About 25% of GCAP-1
is lost during the purification of ROS on a sucrose gradient,
whereas none of GCAP-2 was lost (data not shown) Taking
into account the loss of GCAP-1 the ratio of GCAP-1 to
rhodopsin is 1 : 900 These values correspond to a cellular concentration of 3.3 lM GCAP-1 and 2.7 lM GCAP-2 Thus, both GCAPs are present in bovine rods in nearly equal concentrations
Catalytic efficiency of ROS-GC1 Previous determinations of the ratio of ROS-GC1 to rhodopsin in bovine ROS, based on the purification of ROS-GC1 from ROS preparations ranged from 1 : 104 [6]
to 1 : 440 [9] We re-examined the amount of ROS-GC1 by
a different approach using the chemiluminescent densito-metric analysis of blot membranes as described above for the GCAPs For example, ROS with 2 lg of rhodopsin
Fig 1 Quantitation of GCAPs in purified bovine ROS Increasing amounts of purified GCAP-1 (A: a, 0.5 ng; b, 1 ng; c, 2 ng; d, 3 ng; e,
4 ng; f, 5 ng) and GCAP-2 (B: a, 2 ng; b, 3 ng; c, 4 ng; d, 5 ng; e, 6 ng;
f, 7 ng; g, 8 ng; h, 9 ng) were used to create a calibration curve The two GCAP-1 bands visible in the calibration row are nonmyristoylated and myristoylated GCAP-1 Protein samples were electrophoresed and blotted together with a sample of ROS containing 5 lg (A) or 10 lg (B) of rhodopsin GCAPs in these samples were detected by specific antibodies and ECL Band intensity (arbitrary units) was linear within the tested range, ROS with 5 lg rhodopsin contained 1.8 ng of
GCAP-1 corresponding to a ratio of GCAP-1 : GCAP-1600 GCAP-GCAP-1 to rhodopsin, ROS with 15 lg rhodopsin contained 6.5 ng of GCAP-2 corresponding to a ratio of 1 : 1340 GCAP-2 to rhodopsin.
Trang 4contained 28 ng of ROS-GC1 (not shown) This relates to a
molar ratio of 1 : 200 ROS-GC1 to rhodopsin Analyzing
four different blots revealed a mean ratio of 1 : 260 ± 60,
which is consistent with previous estimates [6,9] The
functional unit of ROS-GC1 is a dimer [16,42,43] that is
correspondingly present in a ratio to rhodopsin of 1 : 520
The cellular concentration of the ROS-GC1 dimer therefore
is 5.8 lM(cellular concentration of rhodopsin¼ 3 mM) We
used this value to calculate the turnover number of
ROS-GC1: Vmax in washed ROS membranes was 3.0 nmol
cGMPÆmin)1per mg Rh, which is synthesized by
4.8· 10)2nmol ROS-GC1 dimer (1 mg of Rh¼ 25 nmol
Rh; ratio of ROS-GC1 to Rh is 1 : 520) Thus, the resulting
turnover number or kcatof ROS-GC1 in native membranes is
1.0 s)1, which is very similar to previous determinations of
the purified enzyme in detergent (0.2–1.3 s)1; [6]) From these
numbers we derive the catalytic efficiency expressed as kcat/
Kmas 0.77· 103
M )1Æs)1(Km¼ 1.3 mM, Table 1) Further-more, our data show that the sum of the molar
concentra-tions of GCAP-1 and GCAP-2 equals the molar
concentration of one ROS-GC1 dimer
GCAP-1 and GCAP-2 influence the catalytic efficiency
of ROS-GC1 in different ways
Equal amounts of GCAPs in bovine rods provoke the
question, why cells express equal levels of protein isoforms
with similar properties It is reasonable to suggest that they differ in regulatory features We tested this assumption by testing two key aspects of ROS-GC1 regulation, change in catalytic efficiency and Ca2+-sensitivity
First, how does the interaction with 1 and
GCAP-2 influence kcat/Km of ROS-GC1? ROS-GC1 in washed ROS membranes was reconstituted with a constant amount
of myr- and nonmyr-forms of GCAP-1 or GCAP-2 As the half-maximal activation (EC50) of ROS-GC1 by GCAP-1 and GCAP-2 occurs well below 1 lM [37], we chose a saturating concentration of 2 lMfor all experiments The ROS-GC1 activity was measured as a function of [Mg-GTP] at a constant free [Ca2+] of 5 nM The catalytic parameters were then determined from a Lineweaver–Burk plot (Fig 2A–D) The results are listed in Table 1 The values of kcat were calculated from the values of Vmax
(expressed as nmol)1Æ min)1per mg Rh) assuming a ratio of ROS-GC1 dimer to Rh of 1 : 520 as described above Myr-GCAP-1 increased the catalytic efficiency of ROS-GC1 about 25-fold (kcat¼ 5.9 s)1; kcat/Km¼ 19.5 · 103
M )1Æs)1) This increase in catalytic efficiency was significantly less pronounced (3.4-fold) when GCAP-1 was not myristoyl-ated, the kcat/Km value was 2.6· 103
M )1Æs)1 When the effect of GCAP-2 was assayed in the same manner the results were different Both GCAP-2 forms, myristoylated and nonmyristoylated, increased Vmaxand decreased Kmto
a similar degree, i.e., for GCAP-2, the influence of the
Table 1 Catalytic parameters of GCAP-dependent activation of ROS-GC1 in washed ROS membranes Assays were performed at low [Ca2+] (2 m M
EGTA).
V max (nmolÆmin)1per mg Rh) K m (m M ) k cat (s)1) k cat /K m ( 103M )1 Æs)1) n
Fig 2 Lineweaver-Burk plots of
GCAP-dependent regulation of GC1 (A)
ROS-GC1 reconstituted with 2 l M myristoylated
D6S-GCAP-1 Reciprocal substrate GTP
concentration 1/[S] is given in m M )1 Plot was
linearized by using the apparent Hill
coefficient n ¼ 1.68 (Table 1) to yield 1/[S] n
Reciprocal activity 1/V is expressed as
nmol)1Æmin per mg Rh Kinetic analysis was
performed in the same manner with 2 l M
nonmyryistoylated GCAP-1 (B), 2 l M
myris-toylated GCAP-2 (C) and 2 l M
nonmyris-toylated GCAP-2 (D) Data points represent
mean of triplicates ± SD Details are given in
(A) and in Table 1.
Trang 5myristoyl group was not very pronounced Myr-GCAP-2
increased the catalytic efficiency kcat/Kmby 13-fold, while
nonmyristoylated GCAP-2 increased kcat/Km by 10-fold
Thus, myristoylation plays a significant role in the ability of
GCAP1, but not of GCAP2, to activate ROS-GC1
Ca2+-sensitivities of GCAP-1 and GCAP-2
Second, do GCAPs differ in their Ca2+-sensitive regulation
of ROS-GC1? To answer this question, we performed
experiments with ROS membranes at different free [Ca2+]
and 2 lMof D6S-GCAP-1 or GCAP-2 A typical result is
shown in Fig 3A,B A striking difference was observed at
the free [Ca2+] at which activation of the cyclase is
half-maximal The IC50 value obtained with GCAP-2 was
significantly lower than that obtained with D6S-GCAP-1
The results were reproducible with different preparations of
bovine ROS and GCAPs: the dose–response curve was
always shifted to lower [Ca2+] with GCAP-2 The IC50
value for Ca2+determined from two to three independent
titration curves for GCAP-2 was 100 ± 32 nMand that for
D6S-GCAP-1, was 707 ± 122 nM The dose–response
curves were, in both cases, cooperative with Hill coefficients
of n¼ 1.46 ± 0.11 and n ¼ 2.4 ± 0.0 for GCAP-1 and
GCAP-2, respectively These different Ca2+-sensitivities of
D6S-GCAP-1 and GCAP-2 were independent of GCAP
concentrations, as similar activation profiles were obtained
1 lMand 10 lMGCAPs
Next, we compared the Ca2+-dependency of the
ROS-GC1 activity in whole ROS and washed ROS membranes in
the presence of both D6S-GCAP-1 and GCAP-2 at a ratio
of 1 : 1 (Fig 3C) that corresponds to the cellular ratio in
bovine rods The IC50values were similar for both curves
(138 nM for whole ROS and 284 nM for reconstituted
membranes) Cooperativity was slightly higher in
incuba-tions with native ROS Interestingly, the significant
inhibi-tion of ROS-GC1 by GCAP-2 at high [Ca2+] (Fig 3B) was
not seen in whole ROS and was less pronounced in
reconstituted membranes, which indicated a compensatory
effect by D6S-GCAP-1 (or native GCAP-1 in whole ROS)
Thus, reconstitution of ROS membranes with GCAPs at
physiological concentrations can reproduce the activation
profile of ROS-GC1 in native ROS We conclude from
these results that GCAP-1 and GCAP-2 confer different
Ca2+-sensitivities to ROS-GC1 in bovine rods
We then proceeded to study systems of heterologously
expressed ROS-GC1 and purified GCAPs to answer the
question whether the reconstitution of the native activation
profile requires components specific to native ROS
mem-brane preparations or not Thus, ROS-GC1 was
hetero-logously expressed in HEK293 tsA cells, HEK293 cells and
COS cells Cell membranes were reconstituted with D6
S-GCAP-1 and GCAP-2 and the EC50and IC50values were
determined EC50 values were similar as described
previ-ously [25,44] and maximal activity at saturating GCAP
concentration was similar (data not shown) When we
analyzed the activation profile as a function of free [Ca2+],
we obtained for ROS-GC1 that was heterologously
expressed in HEK293 or HEK tsA cells, similar results as
with native ROS-GC1: GCAP-1 and GCAP-2 differed in
their Ca2+-sensitivities Figure 4A shows a typical series of
experiments with HEK tsA cells The determined IC
values were 609 nMfor GCAP-1 and 47 nMfor GCAP-2 However, a difference in the IC50values was not observed when ROS-GC1 was expressed in COS cells (Fig 4B) We observed an IC50around 100 nMfor both GCAPs It could
be that one factor or component is present both in native
Fig 3 ROS-GC1 activity in bovine ROS membranes as a function of free [Ca2+] Membranes were incubated with a constant amount (2 l M ) of (A) D 6
S-GCAP-1 (d) or (B) GCAP-2 (s) Crosses (·) are control determinations without added GCAPs These data were averaged from at least three experiments The activity unit is nmol cGMPÆmin)1per mg rhodopsin The data were fitted by the modified Hill equation; V/V max ¼ –Z[Ca 2+ ] n /([Ca 2+ ] n + K n
m ) + 1; V is the activity of ROS-GC1, V max is the maximal activity of ROS-GC1, n is the Hill cooperativity, K m corresponds to IC 50 of Ca 2+ -dependent ROS-GC1 activity, and Z is a constant taking into account that ROS-GC1 activity is not zero at high free [Ca2+] IC 50 ¼ 627 n M for
D 6 S-GCAP-1 and IC 50 ¼ 123 n M for GCAP-2 (C) Regulation of ROS-GC1 in bovine ROS membranes by the combined action of GCAP-1 and GCAP-2 (m) Washed ROS membranes were reconsti-tuted with 1 l M D6S-GCAP-1 and GCAP-2 and the Ca2+-dependent activ ation profile is compared with data obtained with a nativ e ROS preparation (h) Washed ROS membranes did not exhibit any Ca2+ -sensitive guanylate cyclase activity (·).
Trang 6ROS and HEK cells yet it is missing in COS cells The
dependency on the membrane source for ROS-GC1 could
also indicate that cyclase lacks some modification when it is
expressed in COS cells for instance and this modification
could be necessary to exert different actions of GCAP-1 or
GCAP-2, but we have no experimental proof for this
speculation
From the results obtained with native membranes, the
native reconstituted membranes and with the reconstituted
heterologous expression systems of HEK tsa and HEK 293
cells, we conclude that the differences in the Ca2+-sensitive
regulation of ROS-GC1 by GCAP-1 and GCAP-2 are an
intrinsic property of the ROS-GC1/GCAP complex The
effect does not particularly depend on the presence of ROS
membranes and therefore does not require a specific
component found exclusively in ROS
Discussion
A key finding of this study is that the cellular concentration
of GCAP-1 and GCAP-2 approximately sums to the
cellular concentration of a functional ROS-GC1 dimer
(about 6 lM) As the apparent affinities of GCAPs for
native ROS-GC1 are very similar [37], one molecule of
GCAP-1 or GCAP-2 could assemble with one ROS-GC1
dimer and form a functional unit This would lead to two
different ROS-GC1 complexes, i.e., ROS-GC1/GCAP-1
and ROS-GC1/GCAP-2 Alternatively, one ROS-GC1 dimer could assemble with one 1 and one
GCAP-2 leaving a population of ROS-GC1 dimers uncomplexed with GCAPs An experimental distinction between these possibilities is beyond the scope of this study and will require further examination However, our results establish that GCAP-1 and GCAP-2 act on ROS-GC1 in different ways This conclusion is further supported by the following two observations
First, the catalytic efficiency of ROS-GC1 depends on the myristoylation of its regulatory proteins GCAP-1 and GCAP-2 However, both proteins differ in this aspect, as the myristoyl group has a stronger impact in the case of
GCAP-1 So far, it is unclear by which mechanism the myristoyl group contributes to the interaction between both GCAPs and ROS-GC1 The fact that there is a differential impact, it implies that GCAP-1 and GCAP-2 have different target sites in ROS-GC1 Indeed, it has been shown that the target sites of GCAP-1 and GCAP-2 on ROS-GC1 do not overlap [44,45] Furthermore, Hwang and Koch reported recently, that the myristoyl group controls the Ca2+-sensitivity of GCAP-1, but not that of GCAP-2 [36] We emphasize, that our results add a new aspect to the complex regulatory features of ROS-GC1: the myristoyl group makes the GCAP-dependent increase in catalytic efficiency more powerful and this effect is more pronounced in the case of GCAP-1 than in the case of GCAP-2
A second important aspect of our results concerning the differential regulation of ROS-GC1 activity by GCAP-1 and GCAP-2 is that the two GCAPs are sensitive to different levels of free [Ca2+] The detection level of GCAP-2 is one order of magnitude lower than that of GCAP-1 However, at intermediate levels, both GCAPs can detect the changes in Ca2+ intensity Although the Ca2+ IC50 values of ROS-GC1 regulation reported in the literature vary over a large range [4], these differences have not been addressed as specific distinct properties of GCAP-1 and GCAP-2 On the contrary, it has been shown that the Ca2+-dependent activation profiles of GCAP-1 and GCAP-2 coincide (Fig 8C of [21]) Only occasionally have differences between GCAP-1 and GCAP-2 activation profiles been noted but not explicitly discussed [36,46] Therefore, we investigated this problem in a systematic manner by using different membrane systems as a source for ROS-GC1, i.e., native ROS membranes, HEK293tsA, HEK293 and COS cells
In addition we varied the concentration of GCAPs, when testing the activation profiles Different Ca2+-sensitivities
of GCAP-1 and GCAP-2 were observed with all mem-brane systems except COS cells Thus, we conclude, that a differential Ca2+-sensitivity is an important intrinsic property of the ROS-GC1/GCAP complex
One reason, why different Ca2+-sensitivities for GCAPs have been overlooked previously, could be that they are still
in a very narrow, yet physiological range of free [Ca2+] Therefore, it was vital for the interpretation of our results that we determined the free [Ca2+] of our buffer solutions with a Ca2+-sensitive microelectrode
Taking into account that GCAP-1 and GCAP-2 target different sites in ROS-GC1, we hypothesize that regulation
of ROS-GC1 is switched from a GCAP-1 mode to a
GCAP-2 mode or vice versa Shortly after illumination,
Fig 4 Activity of heterologously expressed ROS-GC1 that was
reconstituted with GCAPs (A) Activity of ROS-GC1 expressed in
HEK293 tsA cells as a function of free [Ca2+] at a constant amount of
either D6S-GCAP-1 (d) or GCAP-2 (s) IC 50 values were 609 n M
(D 6 S-GCAP-1) and 47 n M (GCAP-2) (B) Activity of ROS-GC1
expresssed in COS cells as a function of free [Ca2+] at a constant
amount of either 4 l M D6S-GCAP-1 (d) or 15 l M GCAP-2 (s) IC 50
values were 109 n M (D 6 S-GCAP-1) and 111 n M (GCAP-2).
Trang 7when the free [Ca2+] begins to fall, GCAP-1 becomes
active When free [Ca2+] further decreases, ROS-GC1 is
switched to the GCAP-2 mode As it is expected, that
under constant background light the free [Ca2+] reaches a
new steady state value [29], these two modes could also
operate at different background light intensities depending
on the free [Ca2+]
Our results may help to explain the findings of a recent
study on transgenic mice by Howes et al [33] These authors
showed that expression of GCAP-2 in GCAP null mice led
to a phenotype that is clearly different from the wild-type
[34] Flash responses from rods of transgenic mice differed
from wild-type responses mainly in two aspects: the fast
recovery shortly after the maximum response amplitude was
missing and in some cases the flash response ended in a
prominent undershoot The first observation is consistent
with a lack of GCAP-1 that would activate cyclase very
early after a single flash or when the free [Ca2+] had just
begun to fall The observed undershoot indicates a delayed
activation of cyclase by GCAP-2 This is also consistent
with our result, that GCAP-2 is activated at lower free
[Ca2+] than GCAP-1 The operating range of free [Ca2+]
for GCAP-2 will be reached later after flash illumination
On the other hand, expression of GCAP-1 can fully reverse
the observed effect of the lack of GCAP-1 and GCAP-2
This observation can be explained by our results, that
GCAP-1 activates ROS-GC1 over a larger range of free
[Ca2+], at higher free [Ca2+] and therefore with an onset far
earlier in the timeframe of a single-flash response than
GCAP-2 does GCAP-1 can therefore compensate the lack
of GCAP-2
In summary, we have found that both GCAPs are
necessary to restore the native [Ca2+]-dependent activity
profile of ROS-GC1 Their combined presence in a complex
with the catalytic ROS-GC1 dimer may serve to extend the
working range of the Ca2+-feedback on ROS-GC1 and
therefore extend the dynamic range of cyclase regulation
under varying light regimes
Acknowledgements
This study was supported by the Deutsche Forschungsgemeinschaft
(Ko948/5–3) and by USPHS awards EY10828, DC 005349 and
HL58151.
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