Introduction

Top Abstract Introduction Materials and Methods Results Discussion Appendix I Appendix II References

A classic pharmacologic method of receptor and agonist classification is through the use of agonist potency ratios. Under null conditions, the relative potency of agonists is independent of the host cell for the receptor and depends only on the relative affinity and intrinsic efficacy of the agonists for the receptor type (see Appendix I). Deviations in agonist potency ratios therefore are used as presumptive evidence for differences in receptors. This technique is based on the tacit assumption that the mechanism of response production for the agonists involved is the same (i.e., that they produce the same active state of the receptor that then goes on to activate the stimulus-response mechanisms of the tissue host in a uniform manner). Therefore, differences in agonist potency ratios, in contrast to furnishing evidence for differences in receptor types, may alternatively indicate lack of adherence to this tacit assumption. This article describes the induction of variation in agonist relative potency ratios in different tissue host cells for a single transfected receptor, the hCTR2. This raises the possibility that this effect indicates the production of agonist-specific receptor active states by the different agonists.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Appendix I Appendix II References

Molecular Biology and Generation of Stable Cell Lines

Full-length bovine Gs (short form) cDNA was kindly provided by Dr. Pat Casey at Duke University (Robishaw et al., 1986). The full-length cDNAs for mouse Gq (Strathmann and Simon, 1990), rat Go1 (Jones and Reed, 1987), and rat Gi1 (Jones and Reed, 1987) were provided by Steve Rees at GlaxoWellcome, UK. The Nco/HindIII fragment of pT7-5/Gs, BamHI fragment of pSG5/Gq, HincII fragment of pT7-5/Go, and the HincII fragment of pT7-5/Gi1 were isolated and subcloned into pCIN expression vector (Rees et al., 1996). The full-length calcitonin receptor was isolated and subcloned into expression vector pcDNA3 as described previously (Chen et al., 1997).

The expression vectors containing different G proteins were then transfected into HEK 293 cells using calcium phosphate method (Promega, Madison, WI). On day 3 after transfection, the cells were selected using G418-supplemented media at a concentration of 600 µg/ml. After a 2-week selection, colonies were selected and expanded. Stable lines were checked for expression by immunoblotting.

Gel Electrophoresis and Immunoblotting

HEK 293 membrane preparations, made from stable clones containing various amounts of overexpressed G-proteins, were qualitatively evaluated using SDS-polyacrylamide gel electrophoresis according to the procedure of Laemmli (1970). Protein (20 µg) was dissolved in 30 µl of TBS-T buffer (20 mM Tris and 500 mM NaCL, pH 7.5, 0.1% Tween 20) and then incubated with 30 µl of 2× SDS loading buffer containing 5% 2-mercaptoethanol. This mixture was boiled for 5 min and cooled on ice. The denatured protein solution [30 µl (10 µg)] was then loaded into each well of the Novex 10-well, 10% 1.5-mm Tris-glycine gel and run at 120V for 90 to 100 min. The resolved proteins were transferred to Novex nitrocellulose membranes. The nitrocellulose membrane then was incubated in blocking buffer (Megga-Block 1:10 in TBS-T) for 1 h. After washing in TBS-T with 1:100 Megga-Block the membranes were incubated with various G specific antibodies (Santa Cruz Biotech, Santa Cruz, CA) for 1 h. After a second washing, the membranes were incubated with a secondary horseradish peroxidase goat anti-rabbit antibody (1:5000) for 1 h. The blot then was developed using the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Piscataway, NJ). Quantitative analysis of the Western blots was done with the BioRad GS-700 Imaging Densitometer (BioRad, Hercules, CA). Bands were measured and analyzed using the BioRad Molecular Analyst software package and data expressed in RDU. Clones were selected based on the increased density of G protein band compared with control.

Transient Transfection of hCTR2

HEK 293 cells enriched with -subunits Gi, Gs, Go, and Gq were plated at a density of 10⁷ cells/225-cm² flask and grown overnight in DMEM plus 10% fetal calf serum supplemented with L-glutamine (2 mM). Cells were transfected with 40 µg of pcDNA3 vector control (Invitrogen, Carlsbad, CA) or pcDNA3/hCTR2 using the calcium phosphate DNA transfection method (Davis et al., 1986). After a 6-h transfection, media were replaced and cells were grown an additional 48 h when they were collected for assay. HEK 293 cells (wild-type, Gi-21, Gs-24, Gq-15, and Go-13 line) were plated to a concentration of 106 cells/100-mm dish. After 24 h, cells were cotransfected with clone 134/pMTR with pRSV/neo at a ratio of 10:1, respectively, according to the calcium phosphate method (Promega).

Binding Studies

Membrane Preparation. At confluency, cultured cells were harvested by manual scraping of the tissue culture flasks. Cells were pelleted by centrifugation at 2000 rpm for 15 min and then homogenized (three 15-s bursts) in ice-cold HEPES buffer (20 mM HEPES, pH adjusted to 7.4 with NaOH at 23°C). The homogenate was centrifuged at 48,000g for 15 min and washed twice through resuspension with new buffer. After a third centrifugation, the pellet was resuspended in fresh buffer containing 0.2 mM phenylmethylsulfonyl fluoride. Aliquots were frozen at 70°C.

Saturation Binding Curves. Membranes were equilibrated with either ¹²⁵I-AC512 (2000 Ci/mmol; Amersham Pharmacia Biotech) or ¹²⁵I-hCAL (2000 Ci/mmol; Amersham Pharmacia Biotech) in 20 mM HEPES buffer containing 0.5 mg/ml bacitracin, 0.5 mg/ml BSA, and 0.2 mM phenylmethylsulfonyl fluoride (all from Sigma Chemical, St. Louis, MO) for 60 min at 23°C (samples mixed on a Titer Plate Shaker; Lab Line Instruments, Melrose Park, IL). Nonspecific binding was defined as the radioactivity remaining in the presence of 100 nM nonradioactive salmon calcitonin. Incubations were started by addition of membrane in triplicate tubes and binding was terminated by filtration through glass-fiber filters (presoaked 30 min in 0.5% polyethylenimine), with the Skatron semiautomatic cell harvester. Filters were placed in Sarstedt 68.752 51- × 12-mm polypropylene tubes and counted for 1 min in a gamma counter.

Measurement of Calcium Transient Responses

Stable HEK 293 clones were tested for their ability to mobilize calcium in the FLIPR system. Cells were harvested with 0.05% trypsin (Life Technologies, Gaithersburg, MD) and plated in black 96-well Viewplates (Polyfiltronics, Rockland, ME) at a concentration of 10,000 cells/well in DMEM/F12 phenol red free medium (Life Technologies) containing 5% fetal bovine serum (Life Technologies). Approximately 30 h after cell plating, the media was removed by vacuum and replaced with DMEM/Ham's F12 phenol red-free medium without serum. Cells were kept in serum-free medium approximately 18 h before assay. At the time of assay, cells were washed with 100 µl FLIPR buffer (145 mM NaCl, 5 mM KCl, 0.5 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, and 10 mM glucose, pH 7.4). Dye was prepared as follows: 2 mM calcium green stock (C 3011; Molecular Probes, Eugene, OR) was prepared in dimethyl sulfoxide. The stock was mixed 50/50 with 20% pluronic acid (P 3000; Molecular Probes) and diluted to 4 µM final concentration in FLIPR buffer. Cells then were loaded with 50 µl of the 4 µM calcium green dye along with 2.5 mM probenecid in 2.5% NaOH and allowed to incubate for at least 1 to 2 h at 37°C. After incubation, the plates were allowed to come to room temperature. Plates were washed in FLIPR buffer twice and 50 µl left in each well. After loading plates into the FLIPR, basal intracellular calcium [Ca²⁺]_i conditions were monitored for 10 s before adding 50 µl of the agonist with an integrated 96-well pipettor. Fluorescence was measured from all 96 wells simultaneously using a charge-coupled device camera. Agonist activity was measured every second for the first 25 s then every 3 s for the next 15 s. Curves were calculated as percentage of 10 µM ionomycin control response.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Appendix I Appendix II References

Stimulus-Biased HEK 293 Cells. Approximately 100 HEK 293 cell lines were transfected with G-subunit cDNA for four different G proteins (Gi, Gs, Go, and Gq) and made into stable clones. These were subjected to Western blot analysis for visualization of relative quantities of specific G proteins. As shown in Fig. 1, certain clones contained elevated levels of Gi, Gs, Go, and Gq protein, respectively. Accordingly, cell lines 21 (for Gi-enriched cells), 24 (Gs-enriched), 13 (Go-enriched), and 15 (Gq-enriched) were selected for further study. These stable cells lines then were transiently transfected with hCTR2 cDNA for binding and functional studies.

View larger version (51K): [in this window] [in a new window]   Fig. 1.   Western blot gels showing relative amounts of G-protein in the membrane of various colonies of HEK 293 cells transiently stably transfected with cDNA for Gi-, Go-, Gs-, and Gq-. Relative to control, stable colonies 21 (control, 11.26 RDU; Gi-enriched, 23.47 RDU); 13, (control, 2.06 RDU; Go-enriched, 10.41 RDU), 24 (control, 2.05 RDU; Gs-enriched, 8.01 RDU), and 15 (control, 11.26 RDU; Go-enriched, 10.56 RDU) were chosen for receptor cotransfection.

Saturation Binding Studies. Saturation binding curves were obtained for the agonist ¹²⁵I-hCAL and the calcitonin receptor antagonist ¹²⁵I-AC512 (Chen et al., 1997) on membranes prepared from transiently transfected (expressing hCTR2) wild-type HEK 293 cells and G-subunit enriched HEK 293 cells. The maximal density of expressed hCTR2 sites as measured with the antagonist radioligand exceeded the number estimated with ¹²⁵I-hCAL (Table 1) consistent with the idea that the G protein limited the production of high-affinity agonist binding state [i.e., the number of receptors exceeded the available G protein (Chen et al., 1997; Kenakin, 1997b)]. Transfection of the four cell lines led to varying levels of receptor expression being highest in the wild-type and Gi-enriched and lower in Gs-, Go-, and Gq-enriched cell lines as measured with ¹²⁵I-AC512 binding (Table 1). Little change in the amount of high-affinity binding complex with ¹²⁵I-hCAL was obtained with G-subunit enrichment.

Effect of Receptor Density on Agonist Potency Ratios. As demonstrated by the different B_max values for ¹²⁵I-AC512 binding, receptor expression of hCTR2 after transient expression was not uniform. This precluded effective comparison among cell types and focused attention on the important aspect of these studies from the standpoint of receptor theory, namely the internal relative profiles of agonists within each cell type. According to the classic models of GPCRs, differences in receptor density should affect absolute potency but not relative potency; this is described more fully in Appendix I. The possible effect of calcitonin receptor density differences was examined in functional studies. The relative potency of agonists for hCTR2 under two different transfection conditions in HEK 293 cells was explored in a stable high expression cell line (30.05 ± 3 pmol/mg of protein hCTR2; Chen et al., 1997) and transient expression in wild-type HEK 293 cells. Figure 2A shows dose-response curves for rat calcitonin in the two cell lines. As shown in this figure, the maximal response to was greater in the stable HEK 293 cell line. Figure 2B shows the correlation of the potencies of the agonists (quantified as pEC₅₀, the -log of the EC₅₀, concentration producing half-maximal response) in the two cell lines. Linear regressional analysis (Snedecor and Cochran, 1967; Armitage, 1971) indicated a highly significant regressional coefficient (T = 8.29, d.f. = 6, P < .001). Fig. 2C shows a graphical representation of the pEC₅₀ values in the stable and the transient cell line. This representation shows the uniform phase shift in potency (slightly higher in the stable cell line) for the agonists; the pEC₅₀ values are given in Table 2. Although the absolute potencies differed, it is important to note that the relative potency, a parameter dependent only upon the agonists and receptor type (Appendix I), did not.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   The relative potency of hCTR2 agonists in a stable HEK 293 cell line containing hCTR2 (30.05 pmol/mg protein) and in a HEK 293 cell line transiently expressing hCTR2. A, dose response curves to rat calcitonin in stable cells (filled circles) and HEK 293 cells transiently transfected with cDNA for hCTR2 (open circles). B, pEC50 values for agonists in transiently transfected cells (abscissae) and stable cells (ordinates). Values shown for eel calcitonin (), salmon calcitonin (), porcine calcitonin (), chicken CGRP (), human calcitonin (), rat calcitonin (), rat CGRP (), and rat amylin (). C, change in pEC50 from stable to transient cells (ordinates = pEC50). For B and C, bars represent S.E.M.

Stimulus-Biased Assay Systems: Effects of Agonists. The effects of standard agonists for endogenous receptors in wild-type and Gs-subunit enriched HEK 293 cells are shown in Fig. 3. As can be seen from this figure, carbachol produced 150% maximal ionomycin responses in Gs-enriched host cells and only 35% maximum response in Go-enriched cells (Fig. 3A). Similarly, responses to ATP were enhanced in Gs-enriched host cells and eliminated in Go-enriched cells (Fig. 3B). These data indicated that, unlike binding for transfected calcitonin receptors, Gs-, Go-, and Gq-protein enrichment produced differences in stimulus-response coupling. For both carbachol and ATP, little difference was seen with Gi-enriched cells compared with wild-type cells.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Responses to carbachol (A) and ATP (B) agonists for receptors endogenous to HEK 293 cells in wild-type cells (), Gs-enriched cells (), Gi-enriched cells (), and Go-enriched cells (). No satisfactory dose-response relationships could be obtained with Gq-enriched cells. Data shown from one of three experiments.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   hCTR2-mediated responses to amylin (A) and rat calcitonin (B) in wild-type HEK 293 cells (), Gs-enriched cells (), and Gi-enriched (). Data shown from one of three transient transfections.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   The relative potency of hCTR2 agonists in wild-type transiently transfected HEK 293 cells and Gi-enriched cells. A, pEC50 values for agonists in wild-type cells (abscissae) and Gi-enriched cells (ordinates). Values are for eel calcitonin (), salmon calcitonin (), porcine calcitonin (), chicken CGRP (), human calcitonin (), rat calcitonin (), rat CGRP (), and rat amylin (). B, change in pEC50 from stable to transient cells (ordinates = pEC50). For A and B, bars represent S.E.M.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   The relative potency of hCTR2 agonists in wild-type transiently transfected HEK 293 cells and Gs-enriched cells. A, pEC50 values for agonists in wild-type cells (abscissae) and Gs-enriched (ordinates). Values are for eel calcitonin (), salmon calcitonin (), porcine calcitonin (), chicken CGRP (), human calcitonin (), rat calcitonin (), rat CGRP (), and rat amylin (). B, change in pEC50 from stable to transient cells (ordinates = pEC50). For A and B, bars represent S.E.M.

View larger version (30K): [in this window] [in a new window]   Fig. 7.   Dose-response curves for amylin () and rat calcitonin () (A) and amylin () and porcine calcitonin () (B) in wild-type HEK 293 cells (left) and Gs-enriched HEK 293 cells (right). p.r., potency ratios for the agonists.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Appendix I Appendix II References

The observation of varying relative potencies of agonists for the same receptor in different host systems is highly unusual. If this were observed in different natural systems, it would be taken as presumptive evidence for differences in the receptors mediating the responses in those systems. The fact that this was observed for a single receptor in different host cells is particularly interesting in that such behavior cannot be accommodated by the tacit assumption made in classical receptor occupancy theory, namely that all agonists form a single active receptor state that then initiates cellular signaling.

In this study, the measurement of selective G protein enhancement was operational. Although the relative increase in the G protein content of the stimulus-biased cells was measured (relative to wild-type control), the resulting data are not relevant to the actual quantity of G protein accessible to the transfected receptor and available for receptor coupling and served only as a method of choosing cells for receptor transfection. The change in the responses to ligands for endogenous receptors (ATP and carbachol) and transfected hCTR2 was more relevant in that a bias, in terms of G protein coupling to these receptors, was demonstrated. This operational observation of G protein enrichment was used as the basis for further study of relative agonist potencies.

The fact that the amount of high-affinity agonist complex was so much lower than the total receptor number (as measured with the antagonist radioligand) indicated that the amount of G protein was limiting. G-subunit enrichment theoretically could have resulted in differences in the amount of high-affinity binding of an agonist radioligand. In keeping with these predictions, coexpression of secretin receptors with Gs has been shown to lead to an increase in the number of high-affinity binding sites for ¹²⁵I-secretin from 1.8% to 15% (Ishihara et al., 1991). However, for pleiotropic receptors that interact with more than a single G protein (such as hCTR2; Horne et al., 1994), the degree to which high-affinity binding would be enhanced depends upon the relative stoichiometries of the G proteins involved (i.e., if a relatively minor G protein is enhanced in the presence of a high concentration of the major interactant G protein for that receptor, then enrichment of a secondary protein may not be detectable as an increase in the number of high affinity binding sites). The relationship of the B_max for an agonist observed in saturation binding and G protein is given from eq. 7 of Appendix II as L (1 + ₂[G₂]/₂K₂) [R_total]/(1 + L (1 + ₁[G₁]/₁K₁+ ₂[G₂]/₂K₂)). It can be seen that enrichment of one of the G proteins would have little effect on B_max if the other G protein binding the receptor was in a relatively greater concentration or if the affinity of the receptor active state (denoted by ) was lower for the enriched G protein. Thus there is a limitation of mass equivalence in the measurement of G-subunit protein in binding studies.

The limitation in binding studies of mass equivalence is not present in functional studies. When cell function is used as a measure of receptor stimulus, there can be disconnections between the amount of high-affinity complex and resulting amount of stimulus (and resulting response) produced by that complex. In general, stimulus-response mechanisms within cells greatly amplify the result of receptor/G protein interaction therefore G protein enrichment, insufficient to result in changes in high-affinity binding, may still produce observable effects on function. In light of the data with Gs-enrichment, this seems to be what occurred in these studies. The implications of this finding are worth considering.

Within a given cell type, if all agonists produce a uniform active receptor state, then the relative potency of these agonists will not vary (see Appendix I). The data obtained in Gs-enriched cells cannot be accommodated by this hypothesis, leading to the possibility that some of the agonists in this study produce at least two different receptor active states (see below). There are increasing data in the literature, from a range of experimental approaches, to suggest that this occurs with other receptors. Experimental data support the idea that agonist specific receptor activation for PACAP 1 receptor (pituitary adenylate cyclase activating polypeptide receptor type 1; Spengler et al., 1993), Drosophila tyramine receptors (Robb et al., 1994), and 2-adrenoceptors (Chidiac et al., 1994). Agonist specific receptor activation has directly been proposed for dopamine D_2s (Wiens et al., 1998), 5-HT_2c (Berg et al., 1998), 5-HT₃ (Van Hooft and Vijverberg,1996), ₂-adrenoceptors (Krumins and Barber, 1997; Seifert et al., 1999), cannabinoid CB₁ receptors (Bonhaus et al., 1998; Glass and Northup, 1999) and µ-opioid receptors (Keith et al., 1996; Blake et al., 1997; Yu et al., 1997).

Agonist-specific receptor active states have been hypothesized on the basis of a number of experimental approaches. These include observation of protean agonism [reversal of efficacy from positive to negative in quiescent versus constitutively active receptor systems (Kenakin, 1997c)], differences in agonist-induced receptor internalization, kinetic rates of activation, differences in rates of hydrolysis of G protein-induced nucleotide hydrolysis, and reversal of the relative potency of agonists for different stimulus-response pathways. Many of these approaches uncover agonist-selective receptor states, but it is still a theoretical hypothesis that these states are involved in signaling and will result in stimulus-trafficking. The present approach directly demonstrates that the agonists tested produce differing response patterns which may translate to different therapeutic profiles. On the other hand, complex differential G protein coupling would be expected to be dependent upon receptor/G protein stoichiometry and would thus be quite system dependent. Under these circumstances, it would not be expected that a given stimulus-biased assay such as this one would be universally useful for the detection of stimulus trafficking. With this in mind, it would be prudent to use as many techniques as possible to test ligand agonism because some may work better than others for given receptors and agonists.

The ternary complex model for GPCRs, either in the form of the extended ternary complex model (Samama et al., 1993), or the cubic ternary complex model (Weiss et al., 1996a,b,c) commonly are described as "two-state" models referring to the two spontaneously occurring active and inactive states of the receptor. The concept that agonists produce unique active receptor states may seem to be in conflict with these classic models. However, it should be noted that these models really are "multistate" models when describing activation of receptors by ligands. This is because the presence of the ligand opens the possibility of a modified affinity of the activated receptor for G proteins through thermodynamic constants  (extended ternary complex model) or  and  (cubic ternary complex model); see Table 4 and Fig. 8. Thus, the existing models of GPCR function accommodate agonist-specific receptor active conformations.

View larger version (25K): [in this window] [in a new window]   Fig. 8.   Two models of GPCR activation. The extended ternary complex model (Samama et al., 1993) allows interaction of only the active state receptor with G protein. The cubic ternary complex model (Weiss et al., 1996a,b,c) allows interaction of both the active and inactive state with G protein. The binding of ligand allows changes in the receptor affinity for G protein through the factors , , and . This allows for agonist-specific receptor active states within the framework of these models.

The formation of agonist-specific receptor active states becomes important from a cellular signaling point of view when multiple G proteins are involved. It is known that many G protein coupled receptors are pleiotropic with respect to the G proteins with which they interact (Kenakin, 1996). As discussed previously, human calcitonin receptors are among those known to activate Gs, Gi, and Gq (Horne et al.,1994). This could be important for signaling because studies have shown that different regions of the cytosolic loops of seven-transmembrane receptors activate different G proteins (Ikezu et al., 1992; Wade et al., 1999). Under these circumstances, it would not be expected that different overall receptor conformations would expose these different G protein-interacting sequences to signaling mechanisms in an identical manner. These ideas, taken in conjunction, open the theoretical possibility that different active receptor conformations selectively activate different G proteins to direct stimulus to different biochemical pathways in cells. When this occurs, it would be predicted that the relative potencies of agonists producing different active states will vary according to the relative amounts of G protein available for stimulus-response coupling. This is discussed further in Appendix II.

Stimulus-biased assay systems, such as the G-subunit-enriched HEK cells used in this study, are not meant to reflect natural physiology but rather are designed to furnish unique information about agonists. In drug discovery programs designed to find agonists, there usually are two possible targets: a complete mimic of the physiologically endogenous agonist or a mimic of a subset of agonism produced by the physiological agonist. Previously, the latter profile has been achieved solely by finding agonists for receptor subtypes or restriction of pharmacokinetics. The production of selective receptor active states theoretically offers another level of selective agonism. Specifically, if the spectrum of signal transduction initiated by a given agonist could be reduced, then a subset of physiological responses could be produced. If the pleiotropic nature of the endogenous receptor signaling is associated with a concomitant plethora of physiological responses (some of which are not desired in the therapeutic field), then reducing these may lead to a better mating of replacement agonist therapy for pathophysiological disorders. From this standpoint, agonist-selective receptor active states could represent the next effective level of agonist selectivity (Kenakin, 1997a).

Presently the relevance of agonist-specific cellular signaling to therapeutic targeting of synthetic agonists is not clear. The extent to which this idea can be capitalized upon therapeutically is unclear. At the least, however, it allows a method of classifying agonists by measures beyond those based simply on strength of agonism. This latter idea assumes that all agonists produce a uniform receptor active state that goes on to produce physiological response on the basis of stoichiometry and nothing more. The idea of agonist specific receptor active states extends that concept to include the "quality" of efficacy as well as the "quantity" of efficacy in describing the activity of agonists. The use of stimulus-biased host cells for surrogate expression of receptors may be a useful tool in this regard.