Regulation of the Coupling to Different G Proteins of Rat Corticotropin-releasing Factor Receptor Type 1 in Human Embryonic Kidney 293 Cells*

The regulation of G protein activation by the rat cor-ticotropin-releasing factor receptor type 1 (rCRFR1) in human embryonic kidney (HEK)293 (HEK-rCRFR1) cell membranes was studied. Corresponding to a high and low affinity ligand binding site, sauvagine and other peptidic CRFR1 ligands evoked high and low potency responses of G protein activation, differing by 64-fold in their EC 50 values as measured by stimulation of [ 35 S]GTP (cid:1) S binding. Contrary to the low potency response, the high potency response was of lower GTP (cid:1) S affinity, pertussis toxin (PTX)-insensitive, and homologously desensitized. Distinct desensitization was also observed in the adenylate cyclase activity, when its high potency stimulation was abolished and the activity became low potently inhibited by sauvagine. From these results and immunoprecipitation of [ 35 S]GTP (cid:1) S-bound G (cid:2) s and G (cid:2) i subunits it is concluded that the high and low potency [ 35 S]GTP (cid:1) S binding stimulation reflected coupling to G s and G i proteins, respectively, only G s coupling being homologously desensitized. Immunoprecipitation of [ 35 S]GTP (cid:1) S-bound G (cid:2) q/11 revealed addi-tional coupling to G

The hypothalamic peptide corticotropin-releasing factor (CRF) 1 not only regulates the stress response in mammals by activation of the pituitary adrenal axis (1) but is also involved in the control of the immune response, cardiovascular, reproductive, and cognitive function, ingestive behavior, pregnancy and labor (for a review, see Refs. 2 and 3). The multiple actions of CRF are mediated by two classes of specific CRF receptors, CRFR1 (4 -6) and CRFR2 (7,8), which are encoded by unique genes and of which some variants exist, produced by alternative processing of the transcripts from each of the genes (for review, see Ref. 3). Further mammalian endogenous ligands of the receptors, urocortin (9), stresscopin-related peptide/urocortin II (10,11), and stresscopin/urocortin III (10,12), were detected. The different expressions of the CRF receptor types and their ligands in tissues (for review, see Ref. 3) suggest that they are involved differently in the manifold physiological functions of the CRF receptor system.
The CRF receptors belong to the G protein-coupled receptors (GPCRs). So far, CRFR1 and CRFR2 have been shown to couple to G s proteins, leading to the stimulation of adenylate cyclase in native tissues and cells, in various brain-derived and peripheral cell lines, and in cells transfected with the receptors (for review, see Refs. 2 and 3). Additionally, by using the nonhydrolyzable GTP analog [␣-32 P]GTP␥-azidoanilide to label the G proteins when activated by the receptor, followed by immunoprecipitation with specific G protein antibodies, it was shown that the human CRFR1 is able to activate, in addition to G s , also G i and G q in HEK293 and Chinese hamster ovary cells expressing the receptor (13,14) as well as in the rat cerebral cortex (15). From these results second messengers other than cAMP or even inhibition of cAMP levels might be additionally implicated in CRF signaling. Indeed, urocortin was found to activate the G q /phospholipase C/inositol triphosphate/protein kinase C pathway in HEK293 cells expressing the human subtype CRFR1␣ (14).
From the above mentioned findings it is suggested that the CRFR1 adds to the growing list of GPCRs that simultaneously couple to unrelated G proteins and show multiple signaling (16). To come to conclusions on the regulation of the G protein coupling of CRFR1, in this investigation we studied the conditions for the coupling of the CRFR1 to different G protein classes as well as the functional consequences and relations to the receptor activation, using HEK cells stably transfected with the rat receptor as cellular model.
HEK293 Cell Culture and Transfection with rCRFR1-HEK293 cells were maintained at 37°C under 5% CO 2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 IU/ml penicillin, and 100 g/ml streptomycin. For stable expression, HEK293 cells were plated in 60-mm culture dishes at a density of 4 ϫ 10 5 cells/dish, grown overnight, and transfected with cDNA encoding for rCRFR1 in the expression vector pcDNA3, using LipofectAMINE. G418-resistant cells were selected in Dulbecco's modified Eagle's medium containing 400 g/ml G418. Clones of G418-resistant cells were examined for [ 125 I]Tyr 0 -sauvagine binding to detect cells that expressed CRFR1.
Nontransfected and stably transfected HEK293 cells were seeded in 100-mm culture dishes at a density of 1-2 ϫ 10 6 cells/dish and grown at 37°C to about 90% confluence in Dulbecco's modified Eagle's medium, containing additionally 400 g/ml G418 for the stably transfected cells. The cells were harvested 96 h after seeding. In some cases, 100 -200 ng/ml PTX was added to part of the stably transfected cells 24 h before harvesting the cells to inactivate the G i proteins. When desensitization of the receptor was studied, the cells were incubated with 1 M sauvagine for 24 h followed by extensive washing (eight times with cell culture medium over 2 h at 37°C) to allow for total clearance of the ligand.
HEK Cell Membrane Preparation-Cells were washed with and collected by scraping into phosphate-buffered saline (8.1 mM Na 2 HPO 4 , 1.5 mM KH 2 PO 4 , 137 mM NaCl, 2.7 mM KCl, pH 7.4). After centrifugation at 400 ϫ g for 5 min, the cells were suspended in buffer A (20 mM HEPES, pH 7.8, containing 1 mM EDTA and 27% sucrose) and homogenized by a Teflon-glass homogenizer (10 strokes, 750 rpm). The homogenate was centrifuged at 20,000 ϫ g for 10 min, and the membrane pellet was resuspended in buffer B (20 mM HEPES, pH 7.8, 1 mM EDTA) and stored at Ϫ70°C. Protein concentrations were determined according to Bradford (17). Membranes obtained from HEK293 cells stably transfected with rCRFR1 are designated HEK-rCRFR1 cell membranes.
Receptor/G Protein Coupling Estimated by Binding of [ 35 S]GTP␥S to HEK-rCRFR1 Cell Membranes-3-10 g of membrane protein was incubated in triplicate at 25°C with generally 100 pM [ 35 S]GTP␥S in a total volume of 500 l of medium consisting of 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.1 M GDP, 10 mM MgCl 2 , 0.2 mM EGTA, 1 mg/ml bovine serum albumin, and 0.15 mM bacitracin (binding medium) for usually 120 min. The reaction was terminated by filtration through Whatman GF/B filters using a Brandel harvester (Gaithersburg, MD), and the filters were counted for 35 S activity.
Concentration-response curves for the stimulation of [ 35 S]GTP␥S binding by activation of the CRF receptor by the peptide ligands sauvagine, 3-I-Tyr 0 ,Gln 1 -sauvagine, urocortin, urotensin I, r/hCRF, and oCRF were fitted by nonlinear regression using the program PRISM 4 (GraphPad Software, San Diego). From response curves of sauvagine in the absence and presence of the CRFR antagonist ␣-helical CRF(9 -41), Schild plots were constructed to characterize the influence of the antagonist on the ligand-evoked [ 35 S]GTP␥S binding. The affinities and capacities (B max ) of the basal binding of [ 35 S]GTP␥S (in the absence of stimulating ligand) and of the binding maximally stimulated by sauvagine were determined by displacement curves of the tracer binding by unlabeled GTP␥S and fitting the curves by the program KELL 6 (BIO-SOFT, Cambridge, UK). In dissociation experiments the membranes were incubated with [ 35 S]GTP␥S and with and without sauvagine for 90 min at 25°C. After chilling in ice, the membranes were centrifuged, resuspended in fresh medium containing 1 M unlabeled GTP␥S, and incubated at 25°C to initiate the dissociation for different times at 25°C. It was checked that in ice no dissociation at all occurred.
Receptor/G Protein Coupling Estimated by Immunoprecipitation of [ 35 S]GTP␥S-bound G␣ s , G␣ i , and G␣ q Subunits-Essentially, the protocol given in Ref. 18 was followed. About 75 g of HEK-rCRFR1 cell membrane protein was incubated with [ 35 S]GTP␥S in the absence (basal) or presence (stimulated) of 1 M sauvagine in 110 l of the above binding medium containing 1 M GDP at 25°C for 30 min. 10 g/ml saponin was included in the incubations, which increased the stimulation over the basal signal. The incubation was terminated by adding 900 l of ice-cold medium, and the membranes were pelleted by cen-trifugation at 20,000 ϫ g for 6 min. Membrane proteins were solubilized in 50 l of precipitation buffer consisting of 1% (w/v) deoxycholate, 1% (v/v) Triton X-100, 0.5% (w/v) SDS, 150 mM NaCl, 1 mM DTT, 1 mM EDTA, and 10 mM Tris-HCl, pH 7.4, on ice for 1 h. After adding an equal volume of precipitation buffer, insoluble material was removed by centrifugation at 25,000 ϫ g for 15 min. 10 l of the anti-G␣ s , anti-G␣ i3 , or anti-G␣ q/11 subunit antibody was added to 100 l of the supernatant and incubated overnight at 4°C. 100 l of the antibody-G protein reaction mixture was incubated under shaking at 4°C for 4 h with 3.3 mg of protein A-Sepharose CL-4B, which had been swollen and washed with precipitation buffer before. Then the beads were pelleted at 13,000 ϫ g for 1 min, washed with precipitation buffer, mixed with scintillation mixture, and counted for 35 S.
CRFR1 Binding Assay in HEK-rCRFR1 Cell Membranes-Hot [ 125 I]Tyr 0 -sauvagine saturation binding curves were obtained from incubations of the tracer with 7-17 g of membrane protein at 25°C for 2 h in 300 l of medium exactly the same as used in the [ 35 S]GTP␥S binding assay (see above). To cover the wide ligand concentration range from about 5 ϫ 10 Ϫ12 up to 2 ϫ 10 Ϫ7 M, two tracer stock solutions were prepared in which the specific radioactivities were reduced from originally 2,200 Ci/mmol to 5-46 and 243-361 Ci/mmol with unlabeled 3-I-Tyr 0 ,Gln 1 -sauvagine. In five experiments, each eight experimental points were determined in the low and high ligand concentration range using the high and low activity tracer, respectively. Nonspecific binding for all tracer concentrations was determined by adding 1 M 3-I-Tyr 0 ,Gln 1 -sauvagine. The samples were filtered through GF/C filters (Whatman) in a Brandel harvester. From the data of the two separate binding curves a common curve was calculated and fitted by the program KELL 6.
Adenylate Cyclase Activity in HEK-rCRFR1 Cell Membranes-About 20 g of membrane protein from HEK-rCRFR1 cells was incubated in duplicate in a reaction mixture consisting of 50 mM Tris-HCl, pH 7.4, 4 mM MgCl 2 , 2 mM EDTA, 1 mM isobutylmethylxanthine, 1 mM cAMP, 100 M ATP, 10 M GTP, 1 mM DTT, 0.75 Ci [ 32 P]ATP, 5.1 mg/ml phosphocreatine, 1.32 mg/ml creatine phosphokinase, and 12 mg/ml bovine serum albumin in a final volume of 100 l for 20 min at 32°C. Sauvagine at the indicated concentrations was added to determine concentration-response curves. The incubations were stopped by the addition of 500 l of a solution that contained 4 mM ATP, 1.4 mM cAMP, 2% SDS, and 1 mM [ 3 H]cAMP (about 10,000 cpm). [ 32 P]cAMP was isolated by sequential chromatography on columns of Dowex cation exchange resin and aluminum oxide and corrected for [ 3 H]cAMP recovery (19). Concentration-response curves were fitted by nonlinear regression using the program PRISM.
Inositol 1,4,5-Triphosphate Accumulation in HEK-rCRFR1 Cells-The assay was performed according to Ref. 20. Briefly, HEK-rCRFR1 cells (100,000/well) were grown in 24-well plates in culture medium. After preincubation with 74 kBq/ml myo-[2-3 H]inositol for 20 h in the absence and presence of 200 ng/ml PTX, the cells were stimulated with different concentrations of sauvagine for 60 min in culture medium without fetal calf serum containing additionally 10 mM HEPES, 0.5% bovine serum albumin, and 10 mM LiCl. The cells were lysed with 150 l/well 0.1 N NaOH, and subsequently 50 l of 0.2 M formic acid, 1,000 l of 5 mM sodium tetraborate, and 1,000 l 0.5 mM EDTA were added. The lysates were centrifuged, and the supernatants were subjected to anion exchange chromatography on SepPak Vac 3-ml Waters Accel TM Plus QMA cartridges. [ 3 H]Inositol 1,4,5-trisphosphate was eluted with 0.1 M formic acid and 0.4 M ammonium formate and counted. Concentration-response curves were fitted by nonlinear regression using the program PRISM.

Optimum Conditions for rCRFR1-activated [ 35 S]GTP␥S
Binding to HEK-rCRFR1 Cell Membranes-Stimulation by sauvagine of [ 35 S]GTP␥S binding to HEK-rCRFR1 cell membranes, used as a measure of total G protein activation, was systematically optimized by examining the effects of factors known to be critical in GPCR-evoked GTP binding, GDP, MgCl 2 , NaCl, DTT, [ 35 S]GTP␥S, temperature, and time. The optimum incubation conditions for a maximum ratio of stimulated:basal binding and, at the same time, high bound activities, were selected to be 0.1 M GDP, 10 mM MgCl 2 , 100 mM NaCl, and 100 pM [ 35 S]GTP␥S tracer, without DTT, which decreased at low concentrations (0.1-1 mM) selectively the stimulation of binding by sauvagine and at higher concentra-tion basal and stimulated binding uniformly. The influence of DTT is in line with findings showing that disulfide bonds in the extracellular amino-terminal part of the CRFR1 are important for the formation of the active receptor state (21). Basal and stimulated binding increased time-dependently both with halflives of about 30 min at, consequently, constant relative stimulation over basal at 25°C. Under the optimum conditions, during incubation over 2 h about 230 fmol [ 35 S]GTP␥S/mg of protein was bound in the absence of a stimulating CRF agonist, and this amount bound was increased by 80 -160% by the CRF agonists. No stimulation of [ 35 S]GTP␥S binding by sauvagine to membranes obtained from nontransfected HEK cells was observed.

rCRFR1/G Protein Coupling Estimated by CRFR1 Agoniststimulated [ 35 S]GTP␥S Binding to HEK-rCRFR1 Cell Membranes and to G␣ s , G␣ i , and G␣ q Subunits in the Membranes-
Using intact membranes, concentration-response curves of all peptidic CRFR1 agonists studied were clearly biphasic, resulting in two EC 50 values corresponding to high potency and low potency responses around EC 50 (h) 5 ϫ 10 Ϫ11 M and EC 50 (l) 3 ϫ 10 Ϫ9 M, respectively ( Fig. 1 and Table I). On average, low and high potencies differed in their EC 50 values by 64.2 Ϯ 9.3-fold, and the part of high potency response was calculated to be 27.76 Ϯ 1.54% of the maximum activity for the sum of both response phases at 100 pM tracer (from all peptides, n ϭ 21). All agonists stimulated the binding to the same maximum activity (Fig. 2). When the concentration of [ 35 S]GTP␥S was increased from 100 to 1,000 pM, the EC 50 values for sauvagine did not significantly change; however, the part of high potency phase increased from 27.8% to more than 50% (inset in Fig. 1), at a reduced stimulation over basal of 35.2% compared with about 105% at 100 pM tracer.
As expected from the biphasic concentration-response curves obtained with the membranes, CRFR1 did not couple only to G s protein as generally functionally observed. Anti-G␣ s , anti-G␣ i3 , and anti-G␣ q/11 antibodies precipitated solubilized [ 35 S]-GTP␥S-bound protein obtained from the membranes in significantly higher amounts after stimulation with sauvagine as well as compared with native HEK cell membranes (Fig. 4).
Affinity and Dissociation of [ 35 S]GTP␥S Binding to HEK-rCRFR1 Cell Membranes-GTP␥S binding isotherms for basal binding and binding stimulated by sauvagine were fitted according to a one-site and two-site binding model, respectively ( Fig. 5A and Table II). The results show clearly that sauvagine stimulated binding by increasing the apparent affinity of the nucleotide binding site to GTP␥S by more than 10-fold (K d 5 ϫ 10 Ϫ10 M). This increase in affinity should not only be the result of accelerated dissociation of GDP from the binding site but also because of a real increase in affinity of the site because even in the absence of GDP the affinity was increased by receptor stimulation (data not shown). Nevertheless, the nonstimulated binding sites had a rather high affinity (K d about 1 ϫ 10 Ϫ8 M), discriminating them from any nonspecific binding. It must be noted that the parameters given in Table II (including those after pretreatment of the cells with PTX, see below) are, although exact with respect to the models used, rather rough because the continuous displacement curves (Fig. 5) did not allow a clear definition of nonspecific binding for calculations. For this reason, the nonspecific binding had to be tested to give good fits. More realistically, the binding curves seem to reflect a continuum of several binding sites, which is deduced further from the dissociation experiments (Fig. 6). Dissociation of [ 35 S]GTP␥S from basal and stimulated binding sites proceeded similarly, but the basal activity dissociated more rapidly. The curves could not be fitted to simple models, and, furthermore, after a relatively rapid dissociation phase about 70% of occupied sites remained dissociating only very slowly after more than 2 h.
Influence of Pretreatment of HEK-rCRFR1 Cells with PTX on the rCRFR1-activated [ 35 S]GTP␥S Binding to Their Membranes and to Different G␣ Subunits in the Membranes-PTX pretreatment of the cells, known to inactivate G i proteins, totally abolished the low potency response to all peptides seen with untreated cell membranes (Fig. 1), and the curves could only be fitted according to a normal one-site fit. At 100 pM [ 35 S]GTP␥S, the remaining activity comprised 23.9 Ϯ 0.94% (Fig. 2) of that obtained with untreated cells (from three experiments with sauvagine and each one with the other peptides), which closely corresponded to 27.76% activity as found for the portion of high potency response in untreated cells (see above). Furthermore, the EC 50 values closely corresponded to those obtained for the high potency response with untreated cell membranes, being, on average from all peptide curves, 0.55 Ϯ 0.07-fold (for sauvagine from three experiments EC 50 1.63 ϫ 10 Ϫ11 Ϯ 5.74 ϫ 10 Ϫ12 M compared with 3.11 ϫ 10 Ϫ11 M in untreated membranes). These results suggested that the low potency PTX-sensitive phase represented coupling to G i proteins, and the high potency phase coupling to G s . PTX treatment also abolished the sauvagine-evoked increase of G␣ i -[ 35 S]GTP␥S immunoprecipitate, but not that of G␣ s and G␣ q/11 (Fig. 4).
From GTP␥S binding isotherms as shown in Fig. 5 it was  Fig. 1 (untreated cells) were fitted according to a two-site response model, resulting in EC 50 (h) and EC 50 (l) for the high and low potency coupling to G s and G i proteins, respectively. Results are expressed as the mean Ϯ S.E. of more than three independent experiments performed in triplicate.  calculated that the affinity of the stimulated binding sites after PTX pretreatment of the cells was decreased from K d1 5 ϫ 10 Ϫ10 to 1.25 ϫ 10 Ϫ9 M and that the amounts of stimulated binding sites before and after PTX treatment did not significantly differ (B max1 1.90 and 2.07 pmol/mg protein, respectively, Table II). The last fact may be explained by the limitations of the fits of the binding curves (Fig. 5) as discussed above.

Influence of Long Term Incubation of HEK-rCRFR1 Cells with Sauvagine on the rCRFR1-activated [ 35 S]GTP␥S Binding to Their Membranes and to Different G␣ Subunits in the Membranes-Incubation of the cells for 24
h with 1 M sauvagine and extensive washing over 2 h to allow for the washout of the peptide from 1 M to at least 1 pM, totally desensitized the high potency phase of the sauvagine-stimulated [ 35 S]GTP␥S binding but left the low potency G i -coupled phase unchanged (Fig. 7). In this series of experiments (n ϭ 7) the potencies of sauvagine in stimulating monophasically the binding of [ 35 S]GTP␥S to membranes obtained from sauvagine-pretreated cells and in stimulating the low potency binding to membranes obtained from control cells were identical (EC 50 3.47 ϫ 10 Ϫ9 Ϯ 3.54 versus EC 50 (l) 3.70 ϫ 10 Ϫ9 Ϯ 1.32 ϫ 10 Ϫ9 ). The combined treatment of the cells with sauvagine and PTX abolished the whole stimulation (Fig. 7). Sauvagine treatment also significantly diminished sauvagine-evoked increase of G␣ s -and G␣ q/11 -[ 35 S]GTP␥S immunoprecipitate, but not that of G␣ i (Fig. 4). In conclusion, treatment of the cells with PTX, sauvagine, and PTX/sauvagine led to the selective inhibition of the G i -, non-G i -, and total G protein coupling, respectively (Fig. 7).
rCRFR1-activated Changes of Adenylate Cyclase Activity in HEK-rCRFR1 Cell Membranes-Sauvagine was found to stimulate the adenylate cyclase activity in a bell-shaped manner with a maximum reached at 3 ϫ 10 Ϫ9 M sauvagine, independently of whether the cells were pretreated with PTX or not (Fig.  9). However, PTX pretreatment increased the maximum activity by 2.98-fold (Ϯ0.13). Fitting the stimulatory limbs of the curves resulted in nearly equal EC 50 values for untreated and PTX-treated cell membranes (2.36 ϫ 10 Ϫ10 Ϯ 8 ϫ 10 Ϫ11 M and 3.09 ϫ 10 Ϫ10 Ϯ 1.67 ϫ 10 Ϫ10 M, respectively). When the cells were pretreated with 1 M sauvagine for 24 h as described above, no activation of the enzyme was observed, but at concentrations of sauvagine higher than 1 ϫ 10 Ϫ9 the basal activity was decreased (Fig. 9, inset). rCRFR1-activated Stimulation of Inositol Phosphates in HEK-rCRFR1 Cells-As shown in Fig. 10, sauvagine at concentrations higher than 10 nM stimulated the accumulation of inositol phosphates in the cells more than 2.5-fold with EC 50 1.18 ϫ 10 Ϫ7 Ϯ 2.91 ϫ 10 Ϫ8 . Pretreatment of the cells with PTX did not change the potency of sauvagine (EC 50 1.26 ϫ 10 Ϫ7 Ϯ 4.15 ϫ 10 Ϫ8 ) but decreased the maximum stimulation to 47.92 Ϯ 2.29%. DISCUSSION The CRF receptors are generally coupled to G s proteins (13)(14)(15) and stimulate the activity of the adenylate cyclase. However, immunoprecipitation of ␣ subunits of G proteins after their labeling with [␣-32 P]GTP␥-azidoanilide after activation of the CRFR1 led to the conclusion of multiple G protein coupling of CRFR1 (13)(14)(15). This study was aimed at investigating the question of whether the coupling of CRFR1 to different G protein classes, G s , G i , and G q is regulated differently. For this purpose we examined, to our best knowledge for the first time, the functional consequences of CRFR1 activation at almost the earliest receptor-mediated event, by measuring the stimulation of the [ 35 S]GTP␥S binding to the G proteins directly in membranes obtained from HEK293 cells that were stably transfected with the rat CRF receptor type 1 (HEK-rCRFR1 cells).
From the biphasic concentration-response curves for the stimulation of [ 35 S]GTP␥S binding to HEK-rCRFR1 cell membranes (Fig. 1), resulting in two ligand activities different by 64-fold in the EC 50 values (Table I) but equally potently antagonized by the antagonist ␣-helical CRF(9 -41) (Fig. 3, Schild constants 9.2 ϫ 10 Ϫ9 M and 4.8 ϫ 10 Ϫ9 M, respectively), it was concluded that the rCRFR1 in the HEK cells was coupled to two different G protein pools. The high potency phase was clearly shown to represent coupling of CRFR1 to G s for the following reasons. Pretreatment of the cells with PTX, widely used as tool to interfere specifically with the coupling of GPCRs to the members of the family of G i type, left this phase unchanged (Fig. 1), did not change the amount of immunoprecipitated  (Fig. 4), and increased the stimulatory activity of sauvagine on the activity of the adenylate cyclase activity at low ligand concentrations (Fig. 9). In addition, when it was found that long term stimulation of the cells with sauvagine abolished specifically the high potency phase (Fig. 7) it was shown in parallel that the amount of immunoprecipitated G␣ s -bound [ 35 S]GTP␥S stimulated by sauvagine was diminished almost to the level seen with native, nontransfected HEK cell membranes (Fig. 4), and, furthermore, the adenylate cyclase was no longer stimulated (Fig. 9, inset). On the contrary, PTX abolished the low potency phase (Fig. 1) and the stimulation of immunoprecipitated G␣ i -bound [ 35 S]GTP␥S by sauvagine (Fig. 4) totally. Therefore it is concluded that the rCRFR1 is coupled to G i with low ligand potency in addition to G s with high potency (Table I), which should mean that the ligand concentration has a major regulatory function in the coupling of the receptor to different G proteins. Because the portion of G s coupling was found to be enhanced when the [ 35 S]GTP␥S concentration was increased (Fig. 1, inset), it is further concluded that cellular GTP is not only substrate for the G proteins but, in addition to the ligand concentration, also regulates the portions of the different G proteins actually coupled to the receptor. The present results parallel in some respects those found for the activation of the h-5-hydroxytryptamine 1A receptor in Chinese hamster ovary cells where biphasic response curves for the full agonist-stimulated [ 35 S]GTP␥S binding were also found (22). In the latter case, however, the multiple G protein subtypes involved in activation were restricted to the G i class, of which a single subunit G␣ i3 was the sole component involved in the high potency phase.

G␣ s -bound [ 35 S]GTP␥S
The high potency of 3-I-Tyr 0 ,Gln 1 -sauvagine in G s coupling closely corresponded to the high affinity binding sites revealed in the HEK-rCRFR1 cell membranes (Fig. 8B, K d 3.85 ϫ 10 Ϫ11 versus EC 50 (h) 3.24 ϫ 10 Ϫ11 , Table I). However, about 98% of the estimated total receptor sites of 45 pmol/mg were in a very low affinity state, K d 1.47 ϫ 10 Ϫ8 (Fig. 8A), which was close to the potency of Tyr-sauvagine for the low potency stimulation of GTP binding to G i (EC 50 (l) in Table I) and which agreed well with the K d of 3.38 ϫ 10 Ϫ8 M for sauvagine in competing for [ 125 I]Tyr 0 -sauvagine binding in presence of GTP␥S to uncouple the receptor from the G proteins (data not shown). GTP␥S binding isotherms (Fig. 5) showed that the activated receptor stimulated the GTP binding to the G proteins by increasing the affinity of some 2 pmol/mg G s and G i nucleotide binding sites at least 10-fold (Table II). Even within the limitations of the fits of the GTP␥S binding isotherms as discussed under "Results," the existence of about 2 pmol/mg stimulated GTP␥S binding sites as opposed to 45 pmol/mg receptor binding sites seems clearly to indicate that the overwhelming part of receptors were not

total G protein binding) and after pretreatment of the cells with PTX (G s protein binding)
The inhibition curves given in Fig. 5 were fitted according to a one-site and two-site model for the basal and stimulated binding, respectively. Given are the K d (M) and B max (pmol/mg of protein) as the mean Ϯ S.E. from three experiments. K d1 and B max1 refer to the stimulated part of the binding isotherms in presence of sauvagine.  coupled to G proteins. This was in line with the splitting of receptor binding sites into about 98% low and 2% high affinity sites. Taken together it is concluded that the low number of high affinity receptor sites evokes the high potency activation of G s protein, whereas a small number of the low affinity sites couples to G i .
Because the [ 35 S]GTP␥S assay per se does not differentiate among G protein subtypes, CRFR1 coupling to G proteins other than G i and G s could also be involved. Indeed, after receptor activation G␣ q/11 -bound [ 35 S]GTP␥S was immunoprecipitated in higher amounts, which were not significantly lowered after PTX pretreatment of the cells (Fig. 4). In line with these results, sauvagine stimulated the accumulation of inositol phosphates in HEK-rCRFR1 cells (Fig. 10). The ␣ subunits of the G q/11 subfamily have been shown to activate phospholipase C isozymes and to stimulate the inositol phosphate production in a PTX-independent way (23); however, about half of the sauvagine-stimulated production in the HEK-rCRFR1 cells was inhibited after pretreatment of the cells with PTX (Fig. 10). Some receptors were found to activate phospholipase C␤ isozymes also through G ␤ ␥ dimers released from heterotrimer G s or G i proteins (23,24). Obviously, the coupling of CRFR1 in the HEK-rCRFR1 cells to G i proteins leads to the release of ␤␥ subunits that contribute to the stimulation of phospholipase C in a PTX-sensitive manner. This result is contrary to findings showing that G proteins other than G q/11 were not involved in urocortin-induced mitogen-activated protein kinase in HEK-CRFR1 cells, thought to be mediated by the phospholipase C/inositol phosphate signaling pathway (14).
The great differences in ligand potencies for the G s and G i coupling, EC 50 around 5 ϫ 10 Ϫ11 and 3 ϫ 10 Ϫ9 M (Table I) responsible for the different coupling or that the affinities of the G proteins to the activated receptor state differ. The peptidic CRF receptor agonists used stimulated with a potency order as generally known for the CRFR1, urocortin, and oCRF being the most and least potent compounds, respectively (Table  I). All agonists did not differ significantly in their maximum ( Fig. 2) nor in their high and low potency activities, and their ratios of high to low potencies were also identical (Table I). Therefore, all peptides should activate the G s proteins to the same extent as they should do with respect to the G i proteins. In other words, all peptides are likely to use or induce almost the same active receptor states and should not differ in their abilities to stimulate separate stimulus-response pathways via different G protein-coupled messenger systems. This conclusion is not in line with results showing that urocortin stimulated cAMP accumulation in HEK-CRFR1 cells with lower potency than oCRF (25) and that urocortin and CRF regulated differently the GRK3 activity in human retinoblastoma Y79 cells (26) and the mitogen-activated protein kinase signal transduction pathway in human pregnant myometrium and transfected cells (14). The reasons for these different results on agonist-specific trafficking of CRFR1 signaling remain unclear.
The bell-shaped concentration-response curve for the stimulation of adenylate cyclase activity (Fig. 9) was first explained by assuming that the stimulation of the enzyme activity via G s at low sauvagine concentrations became attenuated by inhibition at high peptide concentrations corresponding to the G icoupled phase. After inhibition of G i by PTX the cyclase activity rose 3-fold ( Fig. 9), obviously because of the deactivation of inhibitory G i -coupled activity. However, the bell-shaped response and, therefore, the inhibitory phase remained detectable ( Fig. 9), which means that at least part of the inhibition was not caused by coupling of the receptor to G i . At present there is no explanation for this result. It might be speculated that there is an allosteric site the occupation of which at high agonist concentrations negatively modulates the cyclase stimulation by the orthosteric receptor binding site. Evidence has accumulated that receptor proteins may form dimers especially when overexpressed and that the second binding site to be occupied in the dimer may negatively modulate the response of the active receptor state (27). It could be possible that rCRFR1 in the HEK cells forms dimers, resulting in allosteric inhibition of adenylate cyclase at high ligand concentrations. Indeed, fluorescence resonance energy transfer experiments have shown dimerization of CRFR1 in HEK-rCRFR1 cells 2 ; nevertheless, at present such an explanation remains highly speculative.
One major mode of terminating GPCR signaling is homologous desensitization. Homologous CRFR1 desensitization has already been found in Y79 cells (26,28,29) and in the human neuroblastoma cell line IMR-32 (30). Here, we found that in membranes obtained from HEK-rCRFR1 cells that were stimulated by sauvagine for 24 h, the high potency stimulatory phase in [ 35 S]GTP␥S binding (Fig. 7) was abolished, and the amounts of immunoprecipitated [ 35 S]GTP␥S-bound G␣ s and G␣ q/11 subunits (Fig. 4) were strongly decreased, leaving the G i response (Figs. 4 and 7) nearly unchanged. Furthermore, the adenylate cyclase was no longer stimulated but showed only inhibition of the basal activity at the sauvagine concentration range of G i coupling (Fig. 9, inset). From these results it is concluded that the coupling of CRFR1 to G s and G q but not to G i is desensitized. Unfortunately, it was not possible to estimate the part of G q/11 -bound [ 35 S]GTP␥S contributing to the concentration-response curves as shown in Fig. 1 or Fig. 7.
The mechanism behind the differentiated homologous desensitization of the G protein coupling of CRFR1 remains to be resolved. It has been well established that GPCR kinases (GRKs) play a major role in this process. GRKs phosphorylate serine and threonine residues at intracellular domains of the agonist-activated receptor. This phosphorylation interferes with the G protein coupling of the receptor and promotes the 2 M. Beyermann, manuscript in preparation. interaction of the receptor with intracellular proteins that maintain the inactive state of the receptor and favor its internalization (31,32). Y-79 and IMR-32 cells were shown to response to CRFR1 activation with loss of receptors (28,30). Furthermore, CRFR1 was rapidly phosphorylated in response to high CRF concentration in COS-7 cells (33). This phosphorylation was independent of protein kinase A activation, but in Y-79 cells an up-regulation of GRK3 was observed during desensitization of CRFR1 (26). Therefore, a GRK-mediated mechanism is likely to be involved also in the desensitization of CRFR1. In the last years experimental data have accumulated (34 -36) to suggest that different active conformational states of one and the same receptor exist and may have differing abilities to produce diverse signaling ways. Based on this concept (37), from our results it may be speculated that activation of the CRFR1 in HEK cells results in receptor states that activate G s and G q/11 proteins and are subject in parallel to phosphorylation by a GRK, leading to desensitization, whereas G i proteins are activated by other receptor states the serines/ threonines of which cannot be phosphorylated or if phosphorylated do not interfere with the coupling. In line with this suggestion, there have been already some reports showing that phosphorylation of a receptor through kinases can differently affect the efficiency of coupling of the receptor to different subclasses of G proteins (for review, see Ref. 16). The restriction of desensitization to the stimulatory activities of CRFR1 on the adenylate cyclase may represent a regulatory mechanism that ensures a more rapid decline of the stimulatory effect when at the same time the inhibitory activity, not involved in desensitization, remains unchanged.