Báo cáo khoa học: The properties of phosphodiesterase 11A4 GAF domains are regulated by modifications in its N-terminal domain pptx

The acronym GAF is derived from the proteins in which these domains were initially identified mammalian cGMP-binding PDEs, Anabaena adenylyl cyclases and Keywords adenyl cyclase; cGMP; cy

are regulated by modifications in its N-terminal domain Marco Gross-Langenhoff1, Arnulf Stenzl2, Florian Altenberend1, Anita Schultz1and

Joachim E Schultz1

1 Pharmazeutisches Institut, Universita¨t Tu¨bingen, Germany

2 Urologische Universita¨tsklinik, Universita¨t Tu¨bingen, Germany

The secondary messengers cAMP and cGMP regulate

a variety of signalling pathways in essentially all cells

[1] Their intracellular levels are balanced by the

rates of biosynthesis via adenylyl cyclase (EC 4.6.1.1)

and guanylyl cyclase (EC 4.6.1.2) and of breakdown

via cyclic nucleotide phosphodiesterase (PDEs;

EC 3.1.4.17) The human genome codes for 10

ade-nylyl cyclase (AC) and 21 PDE genes The latter have

been grouped into 11 families based on sequence

simi-larities [2] This set of biosynthetic and degrading

enzymes allows precise regulation of secondary

mes-senger levels in different tissues, in individual cell types

and in different subcellular compartments As far as

PDEs are concerned, all share a highly conserved

cata-lytic domain located C-terminally, yet show consider-able variability in their N-terminal regions where the regulatory domains reside [3,4] PDE1, )2, )4, )5, )6, )10 and )11 have an N-terminal tandem domain arrangement, and this may exert its regulatory control

of the catalytic domains in a mechanistically similar mode PDE2, )5, )6, )10 and )11 have N-terminal tandem GAF domains preceded by N-terminals of different lengths GAF domains are small-molecule-binding domains that have been identified in > 3000 proteins throughout all taxonomic kingdoms [5,6] The acronym GAF is derived from the proteins in which these domains were initially identified (mammalian cGMP-binding PDEs, Anabaena adenylyl cyclases and

Keywords

adenyl cyclase; cGMP; cyclic nucleotide

phosphodiesterase 11; GAF tandem domain;

regulation

Correspondence

J E Schultz, Pharmazeutisches Institut,

Universita¨t Tu¨bingen, Morgenstelle 8,

72076 Tu¨bingen, Germany

Fax: +49 7071 295952

Tel: +49 7071 2974676

E-mail: joachim.schultz@uni-tuebingen.de

(Received 22 October 2007, revised 4

December 2007, accepted 5 February 2008)

doi:10.1111/j.1742-4658.2008.06319.x

The tandem GAF domain of human phosphodiesterase 11A4 (hPDE11A4) requires 72 lm cGMP for half-maximal effective concentration (EC50) of a cyanobacterial adenylyl cyclase used as a reporter enzyme Here we exam-ine whether modifications in the N-terminus of PDE11A4 affect cGMP sig-nalling The N-terminus has two phosphorylation sites for cyclic nucleotide monophosphate-dependent protein kinases (Ser117, Ser168) Phosphoryla-tion of both by cAMP-dependent protein kinase decreased the EC50value for cGMP from 72 to 23 lm Phosphomimetic point mutations (S117D⁄ S167D), which project complete phosphorylation, lowered the

EC50value to 16 lm Structural and sequence data indicate that 196 amino acids precede the start of the GAF domain in hPDE11A4 Removal of 197 amino acids yielded unregulated cyclase activity, whereas truncation by 196 amino acids resulted in a cGMP-regulated protein with a cGMP EC50 value of 7.6 lm Truncation by 176 amino acids was required for cGMP

EC50 values to decrease to below 10 lm; a construct truncated by 168 amino acids had an EC50 value of 224 lm The decrease in EC50 values was accompanied by a sixfold increase in basal activity; the extent of cGMP stimulation remained unaffected, however We conclude that N-ter-minal modifications strongly affect cGMP regulation of hPDE11A4

Abbreviations

AC, adenylyl cyclase; cAK, cAMP-dependent protein kinase; cGK, cGMP-dependent protein kinase; EC 50 , half-maximal effective

concentration; hPDE11A4, human PDE11A4; PDE, cyclic nucleotide phosphodiesterase.

Escherichia coli transcription factor FhlA) [7] cGMP

was found to be a ligand for the GAF domains of

PDE2 [8,9], PDE5 [10], PDE6 [11] and PDE11 [12],

whereas cAMP regulates PDE10 [12]

Here we deal with PDE11A4, a splice variant of the

most recently discovered PDE family [13–16] The four

PDE11 isoforms differ in terms of the length of their

N-terminal domains Only PDE11A4 contains a

com-plete N-terminal tandem GAF ensemble and an

addi-tional 196 amino acid N-terminus [13] Within this

extended N-terminus in vitro phosphorylation sites

have been identified at Ser117 and Ser162 [13] In vivo

phosphorylation and a potential physiological role for

these phosphorylation sites have not been reported

PDE11 is a true dual-substrate PDE, i.e it

hydro-lyses cAMP and cGMP with similar Kmand Vmax

val-ues [13–15,17] The tissue distribution of the PDE11

isozymes has not yet been fully examined It is known

that robust expression in humans occurs in the

pros-tate [18,19], testis [20–22], spermatozoa [22] and brain

[23] The physiological role of PDE11 in the regulation

of cyclic nucleotide levels is currently unclear, partly

because of a lack of any clear pattern in its tissue

dis-tribution and partly because a PDE11-specific inhibitor

has not yet been developed In studies using a PDE11

knockout mouse model, it has been discussed in terms

of its involvement in sperm development and function

[22] In addition, it is speculated that PDE11 plays a

role in the pathology of major depressive disorder [23]

and the development of endocrinal tumours [24]

Fur-thermore, hPDE11 presumably plays a role in various

urological diseases such as benign prostatic syndrome,

erectile dysfunction and premature ejaculation [25,26]

In agreement with the expression of PDE5 and PDE11

in the transitional zone of the prostate, these PDE

iso-forms might also have a role in regulating the

prolifer-ation of glandular epithelial tissue; PDE5 and PDE11

inhibitors might, therefore, prevent the malignant

transformation of prostatic cells

In recent studies, we used a chimera between the

hPDE11 GAF tandem domain and cyanobacterial

ade-nylyl cyclase cyaB1, and identified cGMP as a ligand

for the GAF domain [12] However, 72.5 lm cGMP is

required for half-maximal activation of the AC via the

GAF-A domain of the tandem, a concentration

out-side the physiological range This stimulation depends

on the presence of the hPDE11A4 N-terminus because

a construct in which the N-terminus is replaced by that

of the cyaB1 AC, is not stimulated [12] In another

study with the PDE5 tandem GAF ensemble, we

dem-onstrated that the N-terminus profoundly affects GAF

signalling [27] We therefore probed whether the

196-residue N-terminus which precedes the hPDE11A4

GAF tandem affects intramolecular signalling The data indicate that phosphorylation and⁄ or proteolytic processing of the N-terminus substantially increases cGMP affinity and may be important in hPDE11A4 regulation

Results

Effect of phosphorylation at Ser117 and Ser162 Previously, we have shown that modifications, e.g phosphorylation, within the 148-amino acid N-termi-nus of PDE5 affect intramolecular cGMP signalling toward the cyanobacterial reporter enzyme cyaB1 AC [27] PDE11A4 has an N-terminus of 196 amino acids, i.e other than PDE2 (221 N-terminal residues) it has the longest N-terminus of human GAF-containing PDE2, )5, )6, )10 and )11 With 27 strongly basic amino acids, Arg and Lys, it has a calculated isoelec-tric point of 10.8 and carries 10 positive charges at

pH 7, i.e this N-terminus may be prone to interact with other subdomains For comparison, the N-termini

of PDE2 and PDE5 have isoelectric points of 5.3 and 5.7, respectively The PDE11A4 N-terminus has two phosphorylation sites, Ser117 and Ser162 imbedded in the signature sequences RRA117S for cAMP-dependent protein kinase (cAK; RRXS) and RKA162S for cGMP-dependent protein kinase (cGK; RKXS) It has previously been shown that both sites are phosphory-lated in vitro by cAK or cGK, yet the functional con-sequences have not been reported [13] We used the catalytic subunit of cAK for phosphorylation of the chimeric protein and observed that the EC50for cGMP stimulation of cyaB1 was reduced to 23 ± 1.2 lm, whereas basal activity (7.4 and 6.8 nmoles cAMPÆ

mg)1Æmg)1 for the unphosphorylated and phosphory-lated proteins, respectively) and the extent of stimula-tion were unaffected (n = 2; data not shown) This reduction was due to phosphorylation within the N-terminus because, as we have shown previously, the reporter enzyme AC is not phosphorylated by cAK [27] However, incubation at 30C for 120 min, which was required for phosphorylation, resulted in a consid-erable loss of AC activity, as apparent in the respective control incubations, and precluded further detailed studies Therefore, we used phosphomimetic mutations and replaced both residues with either aspartate or glu-tamate Single mutations of Ser117 (S117D and S117E) or Ser162 (S162D and S1162E) resulted in slight increases in cGMP efficiency which did not reach statistical significance (Table 1) However, in the double-mutant S117D⁄ S162D, cGMP efficacy was enhanced and the EC50 value for cGMP decreased

significantly to 16 lm (Table 1) The extent of cGMP

stimulation was significantly reduced in all but one of

the phosphomimetic mutants compared with

unphos-phorylated wild-type protein The reduction in

maxi-mal cGMP stimulation of the S117D⁄ S162D mutant

was not due to an increase in basal activity (7 nmoles

cAMPÆmg)1Æmg)1) cAMP did not stimulate (data not

shown) Similarly, the Km values for ATP and the

Vmaxvalues were unaffected by the mutations

The PDE11A4 N-terminus affects cGMP

regulation

We have shown that a chimera consisting of the

PDE11A4 tandem GAF domain and the cyaB1

N-ter-minus in front of cyab1 AC, is not stimulated by

cGMP or cAMP [22] This indicated that the

N-termi-nus which precedes the GAF tandem domain either

affects intramolecular signal transduction and⁄ or directly inhibits the C-terminally located AC Based on two available GAF tandem structures and correspond-ingly adapted sequence alignments, we designated Lys196 as the last residue of the N-terminus and the start of GAF-A at Lys197, the position at which a short a helix that is visible in both structures starts [24,28] Accordingly, we generated two shortened chimeras, one starting at Lys197 [PDE11A4(197-568)cyaB1 AC] and one at Lys198 [PDE11A4(198-568)cyaB1 AC] The data corroborated the aforemen-tioned conclusions because PDE11A4(198-568)cyaB1

AC could not be stimulated by cGMP, whereas PDE11A4(197-568)cyaB1 AC was stimulated 3.4-fold

by cGMP with an EC50 value of 7.6 ± 0.9 lm (n = 10), i.e the EC50value was almost 10-fold lower than that for the full-length construct (Fig 1) Km val-ues and Hill coefficients (no cooperativity) for both chimeras were comparable, indicating that AC reporter activity was unaffected Expression of the constructs was comparable, as probed by western blotting of the affinity-purified proteins (Fig 1) We assume that protein folding was impaired in the shortened PDE11A4(198-568)cyaB1 AC chimera

Next, we asked whether the large reduction in cGMP EC50 required complete removal of the N-ter-minus or whether it could be accomplished by less rad-ical shortening A set of N-terminal truncations was planned according to secondary structural predictions (DNA Star Protean) Accordingly, shortened chimeras started with Ser43, Gly110 (prior to the first phosphor-ylation site), Lys119 (just past the first phosphoryla-tion site), Leu149, Leu164 (past the second phosphorylation site), Ala169, Glu177 and Pro187

Table 1 EC 50 values for cGMP and maximal cGMP stimulation for

phosphomimetic mutants in human PDE11A4 tandem GAF domain

constructs.

Chimeric construct

EC50cGMP (l M )

Maximal stimulation (fold)

PDE11 ⁄ cyaB1 (wild-type) 72.5 ± 10.1 3.8 ± 0.4

*P < 0.05 compared with the parent chimera using Dunnett’s

ana-lysis (see text); n = 4–8.

0 40 120

Lys197

–log [cGMP] [ M ]

35 45 66

116 kDa

80 160

Lys198

Fig 1 cGMP dose–response curves of

N-terminal shortened PDE11 ⁄ cyaB chimeras

for cGMP Results are means ± SEM

(n ‡ 4) s, chimera starting at Lys197

[PDE11A4(197-568)cyaB1 AC]; d, chimera

starting with Lys198

[PDE11A4(198-568)cyaB1 AC] In several points the error is

within the limits of the symbol (Right)

Western blots (0.5 lgÆlane)1) of purified

proteins.

Removal of 42, 109, 118, 148, 163 and 168 N-terminal

residues consistently increased basal AC activity

(Fig 2A) and the cGMP concentration needed for

half-maximal activation of the attached cyaB1 AC

remained high or could not be determined precisely

because of low affinity, e.g when 42 or 109 N-terminal

residues were removed (Fig 2B) Basal activity

increased further in constructs starting with Glu177

and Pro187, in conjunction with a distinct increase in

cGMP affinity Thus, removal of at least 176

N-termi-nal amino acids had a dual effect; it released AC from

apparent inhibition by its N-terminus and increased

the cGMP affinity of the GAF tandem domain up to

20-fold (Figs 1 and 2B) Maximal stimulation by

cGMP was significantly affected in three of the nine

truncated constructs (Fig 2C), although the reduction

appeared to be minor By and large, we consider this

to be within the experimental variability for this type

of assay Matters were clearly different with regard to

the EC50 values for cGMP stimulation, as determined

by dose–response curves for all truncated versions In fact, the data did not require statistical analysis (Fig 2C) The EC50 for cGMP stimulation was 3.5 ± 2.2 lm for the construct beginning at Glu177 and 10.2 ± 4.0 lm for the construct beginning at Pro187 (Fig 2C) Thus, only the two latter constructs were comparable with that without an N-terminal region, which had an EC50 value of 7.6 lm cGMP (Fig 1) cAMP did not stimulate the reporter enzyme significantly in any of these constructs (data not shown) cGMP-binding assays were not carried out because the lm affinities precluded promising experi-ments using this technique For all shortened con-structs, the Kmvalues for substrate ATP were found to

be unchanged and all Hill coefficients were below unity (no cooperativity; data not shown) Because the extent

of protein purity after affinity chromatography as eval-uated by SDS⁄ PAGE and western blots was similar, the data were comparable (Fig 3D) The slight SDS⁄ PAGE running inconsistencies (e.g S43, G110)

0

10

30

50

70

Met-1 Ser-43

Gly-110 Lys-119

0

50

100

150

200

Met-1 Ser-43

Gly-110 Lys-119 Leu-149 Leu-164 Ala-169 Glu-177 Pro-187 Lys-197

0

1

2

3

4

Met-1 Ser-43

Gly-110 Lys-119 Leu-149 Leu-164 Ala-169 Lys-197

*

*

*

K-198

116

66

45

35

C

D

Fig 2 The length of the N-terminus of the hPDE11A4 GAF tandem domain affects basal, maximal and cGMP-stimulated cyaB1 AC activity (A) Basal activities, (B) maximal activities with 3 m M cGMP, and (C) EC50values of N-terminally truncated PDE11 ⁄ cyaB AC chimeras The respective starting amino acids are given on the x-axis Values are means ± SEM (n ‡ 4) Asterisks in (B) denote significant differences (P < 0.5) from the full-length chimeras starting at Met1 (D) Western blots of purified recombinant proteins used in (A,B) Detection of proteins was with antibody directed against RGS-His4 against the N-terminal affinity tag Different gels were equalized at the 116-kDa level Molecular mass markers of the individual SDS ⁄ PAGE gels are on the left for each gel, respectively Loading of lanes was uniformly with 0.5 lg.

were not due to proteolysis because the western blot

detected the N-terminal affinity tag and C-terminal

degradation at the catalytic domain would have

resulted in a loss of AC activity We noticed that basal

AC activities correlated reasonably well with maximal

activation which was attainable with cGMP

There-fore, we concluded that intramolecular signalling most

likely was unimpaired in the shortened constructs

Discussion

Previously we reported that the tandem GAF domains

of mammalian PDE2,)5, )10 and )1 functionally

cou-ple to the cyanobacterial AC cyaB1 with retention of

their regulatory potency [12,27,29] Thus, cyaB1 AC is

regulated by cAMP when coupled to the tandem GAF

domain of PDE10, and by cGMP when linked to the

tandem GAF domain of PDE2, )5 or )11 The

bio-chemical properties of signalling by human tandem

GAF domains to cyaB1 AC were by and large in

agree-ment with data obtained in studies using bacterially

expressed GAF domains, e.g the cyclic

nucleotide-binding affinities of PDE2 and)5 GAF domains

How-ever, we were able to determine additional GAF

domain properties because of the dissociation of the

allosteric regulators, cyclic nucleotides and the substrate

of the reporter enzyme, ATP As far as AC regulation

by the PDE11A4 tandem GAF domain is concerned,

we have previously reported that cGMP activates with

an EC50of 72.5 lm [12] Such a cGMP concentration is

reached under only exceptional circumstances, if at all

This may be discussed in two ways: either cGMP

regu-lation of PDE11A4 via its N-terminal tandem GAF

domain is physiologically irrelevant or potential

second-ary modifications of the PDE11A4 tandem GAF will

result in increased cGMP sensitivity Here we examined

the second possibility

Effect of phosphorylations at Ser117 and Ser162

In vitro phosphorylation of Ser117 and Ser162 in the

hPDE11A4 tandem GAF domain has been reported

A physiological effect on enzyme activity has not yet

been reported [13] Here, serine phosphorylation by

the catalytic subunit of cAK, albeit not known

whether at position 117, 162 or both, increased cGMP

affinity about threefold We are not aware of the

stoi-chiometry of phosphorylation Because cyaB1 AC

activity decreased considerably during incubation with

cAK at 37C we used phosphomimetic point

muta-tions at posimuta-tions 117 and 162 to evaluate the effect of

phosphorylation (Table 1) Single-point mutations at

Ser117 to Asp or Glu had no effect, whereas similar

ones at Ser162 consistently led to a slight enhancement

in cGMP efficacy It cannot be excluded that individ-ual phosphorylations at these positions might affect additional properties such as intracellular location and translocations, or changes in the interaction profile with other proteins due to charge neutralization of the strongly basic N-terminus However, phosphomimetic mutations at both positions strongly affected cGMP affinity With both positions mutated to Asp, the EC50 value for cGMP was 16.3 lm compared with 72.5 lm

in the non-mutated chimera Therefore, as in PDE5, phosphorylation at the N-terminus of the PDE11A4 GAF tandem may constitute a mechanism regulating the cGMP affinity of the PDE11A4 tandem GAF domain and thus allosterically affect PDE11 activity

The role of the N-terminus of the PDE11A4 GAF tandem domain

The N-termini that precede the GAF domains in mammalian PDEs are of significant length, 221 amino acids in PDE2, 148 in PDE5, 82 in PDE10 and 196 in PDE11A4, and it is conceivable that they have a func-tion in conjuncfunc-tion with the regulafunc-tion of PDE activ-ity in addition to the phosphorylations in PDE5 and PDE11A4 (see above) [27] Indeed, we have shown that shortening of the PDE5 N-terminus significantly affected intramolecular signalling in chimeras similar

to those used in this study To date, cNMP binding assays using PDE11A4 have been negative and regu-lation by cyclic nucleotides is uncertain [13,15,17], possibly because of the low cGMP affinity of the PDE11A4 GAF domains [12] Here, we explored whether the PDE11A4 N-terminus is involved in mod-ulating cGMP affinity We generated a panel of nine N-terminally shortened PDE11A4 tandem GAF chi-meras according to secondary structure predictions This included the complete removal of 196 amino acids Interestingly, removal of Lys197 which con-stitutes the start of the first a helix of GAF-A completely abrogated intramolecular signalling, func-tionally validating the sequence alignment Removal

of up to 169 amino acids yielded variable results that might be summarized as a modest increase in basal

AC activity and a slight increase in EC50 values for cGMP However, removal of 176 amino acids resulted

in a profound change, basal AC activity was increased

 10-fold and cGMP efficacy was increased  20-fold

to an EC50 value of 3.2 lm, fivefold lower than that observed upon phosphorylation of Ser117 and Ser162 (see above) The kinetic parameters of the attached

AC were not altered by N-terminal shortening One may question whether the biochemical data

obtained with the PDE11A4 tandem GA⁄ cyaB1 AC

chimera yields solid information on PDE11A4

holo-enzyme regulation, particularly as no data concerning

PDE11A4 regulation are available for direct

compari-sons However, similar chimeras consisting of the

cyaB1 AC and the tandem GAF domains of PDE2 or

)5 have demonstrated the validity of the approach

used here as far as EC50 values and other parameters

involved in PDE regulation are concerned [27,29]

Therefore, one may postulate that N-terminal

modifi-cations of the PDE11A4 tandem GAF domains are

required to enable cGMP regulation of PDE11

cata-lytic activity Actually, the terminal region may have

two separate effects: one that directly affects cGMP

affinity via the GAF domains, and a second

compo-nent that acts directly on the catalytic activity To

date, no structure is available for the N-termini of

PDE2,)5, )10 or )11 and it will be interesting to see

whether common structural features exist in the

N-ter-mini of GAF-domain-containing PDEs that might

contribute to intramolecular signalling in a similar

manner Another point merits discussion Irrespective

of phosphomimetic mutations or N-terminal

shorten-ings, PDE11A4 GAF-tandem-mediated activation of

cyaB1 AC was always modest, mostly two- to

three-fold, when compared with the effects of the PDE2,

PDE5 and PDE10 tandem GAF domains One may

ask whether this is physiologically significant because:

(a) a two- to threefold change in the Vmax value will

hasten intracellular adjustment of cAMP or cGMP

levels considerably and thus possibly shorten excited

cell states; (b) regulation of PDE4 isozymes is reported

to involve the phosphorylation of a serine located at

the beginning of the tandem of upstream conserved

regions, which precedes the catalytic domain PDE4

activation by this phosphorylation is about twofold,

i.e in the same order of magnitude as that reported

here for PDE11A4 [4]; and (c) it was observed that

removal of the N-terminal portion in long forms of

PDE4 resulted in an increase in basal PDE activity

[4], a situation not unlike that observed here and also

reported for the PDE5 GAF tandem [27]

Finally, one may ask whether phosphorylation at

Ser117 and Ser162 and N-terminal shortening are a

sin-gle mechanism or two independent control mechanisms

We generated phosphomimetic mutations in the

trun-cated constructs beginning at Ser43 and Lys119 In

these chimeras, the EC50values for cGMP stimulation

were either too high to be determined or > 100 lm

(data not shown) Therefore, it seems that we were

deal-ing with two independent control mechanisms In

sum-mary, the data emphasize the important role that the

N-termini of those PDEs possessing an N-terminal

GAF tandem domain may exert in regulation of PDE activity Obviously, structural information for these extended N-terminal regions would help considerably when discussing the biochemical findings in more detail

Experimental procedures

Recombinant DNAs

The cyaB1 gene (gi: 15553050) was a gift from M Ohmori (University of Tokyo, Japan) and a cDNA clone of hPDE11 (gi: 15128482) was provided by G Quintini (Nycomed, Konstanz, Germany) Throughout, the number-ing of amino acids refers to these genes The hPDE11A41-568 cyaB1386-859chimera [12] served as a template to generate all mutants and the N-terminally shortened constructs Sin-gle- and double-point mutations of PDE11A4 Ser117 and Ser162 were generated by fusion PCR with Pfu DNA poly-merase (Promega, Madison, WI, USA) using corresponding sense and antisense primers (MWG Biotech, Ebersberg, Germany) and nearby restriction sites (KpnI, SacI, StuI and MfeI) N-Terminally shortened constructs were created with respective NdeI sense primers and an MfeI antisense primer

in the expression vector pET16b adding a C-terminal His10-tag To generate PDE11A4 GAF-A(181-370)cyaB1(386-859) corresponding parts were amplified by PCR and cloned into pET16b⁄ pQE30 [27] via BamHI, BglII (GAF-A) and BglII, SmaI (cyaB1 AC), respectively An MRGS-His6-tag was located N-terminally

The hPDE11A4(1-568) construct in pQE60 via 5¢-NcoI and 3¢-BamHI was obtained using hPDE11 as a PCR tem-plate This added a C-terminal GSRSHis6 affinity tag The fidelity of all constructs was verified by double stranded sequencing Primer sequences are available on request All pQE plasmids were obtained from Qiagen (Hilden, Germany)

Expression and purification of recombinant proteins

hPDE11A4⁄ cyaB1 chimeras were expressed and purified as described earlier [12] pQE60 constructs were expressed at

16C at 400 lm isopropyl b-d-thiogalactoside overnight and the pQE80 constructs at 18C and at 1 mm isopropyl b-d-thiogalactoside overnight Harvested bacteria were stored at)80C

Adenylyl cyclase assay

Activity was assayed for 10 min at 37C in 100 lL con-taining 22% glycerol, 50 lg BSA, 50 mm Tris⁄ HCl pH 7.5,

10 mm MgCl2 and 75 lm [32P]ATP[aP] (25 kBq; Hart-mann Analytic, Braunschweig, Germany) [30] We added

2 mm [2,8-3H]cAMP (150 Bq; GE Healthcare, Freiburg,

Germany) was added after stopping the reaction to

deter-mine yield during product isolation The reaction was

started by addition of ATP Substrate conversion was

lim-ited to < 10% to ensure linearity PDE activity was absent

in all affinity-purified recombinant proteins All values are

given as mean ± SE Two-tailed Student’s t-tests were used

for statistical evaluation when necessary

Western blot analysis

Proteins were mixed with sample buffer and subjected to

SDS⁄ PAGE (12.5%) Proteins were blotted onto

poly(vinyli-dene difluoride) membranes and sequentially probed with

antibodies directed against either RGS-His4 or His4 (Qiagen)

and with a 1 : 5000 dilution of a peroxidase-conjugated goat

secondary anti-(mouse IgG) (Dianova, Hamburg, Germany)

Peroxidase detection was carried out with the ECL Plus kit

(Amersham-Pharmacia, Freiburg, Germany) Preferably,

western blots of affinity-purified proteins are depicted

because for the current studies it was necessary to ensure that

the constructs did not contain degraded products which

would affect the cyclase reaction N-Terminally degraded

proteins would not have bound to the Ni2+⁄ nitrilotetra ⁄

acetic acid affinity material C-Terminally truncated proteins

are catalytically inactive, i.e western blots show the extent of

purification of the relevant recombinant protein species

Miscellaneous methods

Total protein concentrations were determined using the

method described by Bradford [31], with BSA as the

stan-dard Data are given as means ± SEM of between four

and eight experiments The statistical evaluation of data

was carried out using the Student’s t-test and multiple

com-parisons by one-way analysis of variance (ANOVA)

fol-lowed by Dunnett’s posterior test using graphpad prism

software, version 4.0 for Windows (GraphPad Software

Inc., San Diego, CA, USA http://www.graphpad.com)

A value of P < 0.05 was considered significant

Acknowledgements

We are grateful to Prof M Wahl, Univeristy of

Tu¨bin-gen, for help with the statistical analysis This study was

supported by the Deutsche Forschungsgemeinschaft

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