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J. Biol. Chem., Vol. 277, Issue 23, 21080-21085, June 7, 2002

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A Highly Effective Dominant Negative _s Construct Containing Mutations That Affect Distinct Functions Inhibits Multiple G_s-coupled Receptor Signaling Pathways^*

Catherine H. Berlot

From the Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520-8026

Received for publication, February 8, 2002, and in revised form, March 27, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

To investigate the subcellular organization of receptor-G protein signaling pathways, a robust dominant negative _s mutant containing substitutions that alter distinct functions was produced and tested for its effects on G_s-coupled receptor activity in HEK-293 cells. Mutations in the 35 loop region, which increase receptor affinity, decrease receptor-mediated activation, and impair activation of adenylyl cyclase, were combined with G226A, which increases affinity for , and A366S, which decreases affinity for GDP. This triple _s mutant can inhibit signaling to G_s from the luteinizing hormone receptor by 97% and from the calcitonin receptor by 100%. In addition, this _s mutant blocks all signaling from the calcitonin receptor to G_q. These results lead to two conclusions about receptor-G protein signaling. First, individual receptors have access to multiple types of G proteins in HEK-293 cell membranes. Second, different G protein  subunits can compete with each other for binding to the same receptor. This dominant negative _s construct will be useful for determining interrelationships among distinct receptor-G protein interactions in a wide variety of cells and tissues.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Stimulation of heterotrimeric G proteins by cell surface receptors activates signaling pathways that mediate specific responses to hormones and neurotransmitters. Cells express a wide variety of G protein-coupled receptors as well as numerous G protein , , and  subunits. Many receptors can activate more than one type of G protein, and the G protein subunits can interact with many different types of receptors. The manner in which signaling specificity is maintained in the midst of this vast range of potential interactions is not well understood. This report investigates the interdependence of distinct signaling pathways activated by receptors with broad G protein specificities using a receptor-sequestering dominant negative G protein  subunit.

Many potential mechanisms could establish that distinct receptor-G protein interactions will be independent of each other. Among these, one possibility is that specific receptor-G protein complexes localize to separate membrane compartments (1, 2). Differential associations with particular proteins or lipids (3, 4) or covalent modifications such as phosphorylation (5) may result in subpopulations of receptors and G proteins that have restricted access to each other. Although G proteins are often expressed at much higher levels than their receptors are (6), there is evidence that different receptors utilize separate pools of G proteins (7, 8). An alternative potential mechanism for isolating distinct receptor-G protein interactions is that receptors utilize separate regions for binding different G proteins. If this is the case, then multiple types of receptor-G protein interaction can occur simultaneously without affecting each other. Localization of G protein-binding sites have indicated that separate receptor regions may specify interactions with distinct G proteins (9, 10).

Dominant negative G protein  subunits can test potential mechanisms for separating the distinct G protein interactions of broad specificity receptors. For instance, if interactions with different G proteins are localized to separate subcellular compartments and/or receptor regions, signaling from a receptor to one type of G protein  subunit will be blocked by a dominant negative version of that  subunit, whereas the other signaling pathways will be unperturbed. Alternatively, if each receptor has access to multiple types of G protein and these different G proteins can compete with each other for receptor binding, then a dominant negative  subunit will block all of the G protein signaling pathways activated by the receptor.

Several dominant negative G protein  subunits have been developed previously, but they inhibit G protein signaling incompletely and therefore are not optimal for investigating receptor-G protein signaling pathways. One dominant negative _s mutant contains three substitutions that disrupt different  subunit functions, but it is extremely unstable (11), which contributes to its inability to inhibit signaling completely (12, 13). Xanthine-binding mutants of _o, ₁₁, and ₁₆ can inhibit signaling of specific G protein-coupled receptor families, but inhibition of receptor-mediated phospholipase C stimulation is incomplete (14, 15).  subunit carboxyl-terminal fragments exhibit dominant negative activity (16), but inhibition of G_s signaling is only partial (13).

This report describes the development of a highly effective dominant negative _s mutant that contains substitutions that alter distinct  subunit functions, each of which should stabilize the receptor-bound, nucleotide-free state of G_s. One set of mutations, located in the 35 loop region, specifically increases receptor affinity and decreases receptor-mediated activation without affecting nucleotide handling (17) and also disrupts activation of adenylyl cyclase (18). G226A increases affinity for (19, 20), and A366S decreases affinity for GDP (21). Although A366S alone causes _s to be thermolabile, _s(35/G226A/A366S), containing all three sets of mutations, is expressed at close to wild-type levels and blocks signaling from the luteinizing hormone receptor to G_s by up to 97%. The effects of _s(35/G226A/A366S) on signaling by the calcitonin receptor to G_s and G_q are tested in transiently transfected HEK-293 cells. The results demonstrate that this dominant negative _s mutant can block multiple G protein signaling pathways, which indicates that each receptor has access to multiple types of G protein and that these G proteins can compete with each other for receptor binding.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Construction of _s Mutant Constructs-- _s mutant constructs in the expression vector pcDNAI/Amp (Invitrogen) were generated from the rat _s cDNA (22) containing the EE epitope (23), which was generated by mutating _s residues DYVPSD (residues 189-194) to EYMPTE. Mutations were generated by oligonucleotide-directed in vitro mutagenesis using the Bio-Rad Muta-Gene kit except for those in the 35 region, which were produced by subcloning mutagenic oligodeoxynucleotide cassettes. Subcloning and mutagenesis procedures were verified by restriction enzyme analysis and DNA sequencing.

Transient Expression and Assays for cAMP Accumulation and Inositol Phosphate Formation-- HEK-293 cells (ATCC, CRL-1573) (10⁶ per 60-mm dish) were transfected with plasmids as described in the figure legends using 10 µl of LipofectAMINE 2000 Reagent (Invitrogen) according to the manufacturer's instructions. 24 h after transfection, the cells were replated in 24-well plates and labeled with either [³H]adenine or [³H]inositol.

After an additional 24 h, intracellular cAMP levels in cells labeled with [³H]adenine and inositol phosphate levels in cells labeled with [³H]inositol were determined as described previously (24). cAMP accumulation was measured in the presence of 3-isobutyl-1-methylxanthine and in the presence or absence of agonist as indicated in the figure legends. Inositol phosphate formation was measured in the presence of 5 mM LiCl and in the presence or absence of agonist as indicated in the figure legends. The cells were cultured and assayed at 37 °C.

To determine EC₅₀ values for stimulation of cAMP or inositol phosphate formation, the observed activity was fitted to Equation 1,

(Eq. 1)

where X is the concentration of agonist; Y is the observed cAMP or inositol phosphate formation; a is the cAMP or inositol phosphate formation observed in the absence of agonist; b is the maximum observed cAMP or inositol phosphate formation; c is the half-maximal effective concentration (EC₅₀) of the agonist; and d is the slope factor.

Transient Expression and Assays for Ligand Binding-- HEK-293 cells (6.25 × 10⁶ per 150-mm dish) were transfected with 0.2 µg/10⁶ cells of plasmid encoding the rat luteinizing hormone receptor in pCIS (25) or 0.04 µg/10⁶ cells of plasmid encoding the rabbit C1a calcitonin receptor in pBKCMV (9) and 2 µg/10⁶ cells of vector (pcDNAI/Amp) using 62.5 µl of LipofectAMINE 2000 Reagent. 24 h after transfection, the cells were replated in 24-well plates. Assays for ligand binding were performed after an additional 24 h.

Receptor numbers per cell were determined by fitting data from saturation binding assays using ¹²⁵I-labeled hCG¹ (26, 27) or sCT (9) to Equation 2,

(Eq. 2)

where X is the concentration of agonist; Y is the specific binding; B_max is the maximum number of binding sites, and K_d is the equilibrium dissociation constant. In each case, the medium was removed, and the cells were washed once with binding medium (20 mM HEPES-buffered minimal essential medium with Earle's salts without bicarbonate containing 1 mg/ml bovine serum albumin). The medium was then removed and replaced with binding medium containing 0.062-15 nM ¹²⁵I-labeled hCG in the presence or absence of 0.44 µM unlabeled hCG or 0.030-15 nM ¹²⁵I-labeled sCT in the presence or absence of 1 µM unlabeled sCT. The cells were incubated for 1 h at room temperature, washed three times with ice-cold phosphate-buffered saline, and solubilized with 0.5 N NaOH. The samples were collected and counted in a gamma counter.

Membrane Preparations and Immunoblots-- HEK-293 cells (6.25 × 10⁶ per 150-mm dish) were transfected with 37.5 µg of plasmid using DEAE-dextran (28). 48 h after transfection, membranes were prepared as described (24). 25 µg of membrane proteins were resolved by SDS-PAGE (10%), transferred to nitrocellulose, and probed with a monoclonal antibody to the EE epitope (23). The antigen-antibody complexes were detected using an anti-mouse horseradish peroxidase-linked antibody according to the ECL Western blotting protocol (Amersham Biosciences).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Combining Substitutions in the 35 Loop Region of _s with Mutations That Alter and GDP Binding Results in Highly Effective Dominant Negative Activity-- Replacing five _s residues in the 3 helix and the 35 loop (29) with the homologous _i2 residues (N271K, K274D, R280K, T284D, and I285T) results in an _s construct, _s(35), that exhibits increased affinity for and a decreased ability to be activated by the ₂-adrenergic receptor (17). Independently, the mutations also disrupt activation of adenylyl cyclase by _s (18). These properties suggested that _s(35) might be able to sequester G_s-coupled receptors and exhibit dominant negative activity. Indeed, when _s(35) was transiently expressed in HEK-293 cells that were co-transfected with plasmid encoding the luteinizing hormone receptor, cAMP accumulation in response to 20 ng/ml hCG was inhibited by 42% (S.E. = 3%, n = 3) (Fig. 1).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Combining mutations in the 35 region with mutations that inhibit dissociation and increase GDP dissociation results in an s mutant that can block receptor signaling by 97%. For each data point, 106 HEK-293 cells were transfected with 0.2 µg of plasmid encoding the rat luteinizing hormone receptor in pCIS (25) and 2 µg of vector (pcDNAI/Amp) or plasmid encoding one of the indicated constructs. The average receptor number per cell was 8,400 (S.E. = 370, n = 3). cAMP accumulation was measured in the absence (dark gray bars) or presence (light gray bars) of 20 ng/ml hCG (CR-127, National Hormone and Peptide Program). All values represent the mean ± S.E. of three independent experiments performed in triplicate.

With the goal of producing more effective dominant negative activity, additional mutations predicted to stabilize the receptor-G protein complex were introduced into _s in combination with the 35 substitutions. The effects of adding each of three mutations, G226A, E268A, and A366S, to _s(35) were tested. When combined, these three mutations produce partial dominant negative activity in _s (11). G226A impairs activating conformational changes in switch II required for dissociation of _s from (19, 20). E268A disrupts a salt bridge with Arg-231 that may stabilize the activated conformation of _s (30). A366S elevates basal GDP release, causing _s to be constitutively activated and to spend more time in the empty state (21). _s(G226A/E268A/A366S) inhibited cAMP accumulation in response to 20 ng/ml hCG by 69% (S.E. = 1%, n = 3) (Fig. 1).

When introduced into _s(35), both G226A and A366S produced increased dominant negative activity (Fig. 1). _s(35/G226A) inhibited the cAMP response to 20 ng/ml hCG by 74% (S.E. = 2%, n = 3), whereas _s(35/A366S) inhibited the response by 61% (S.E. = 2%, n = 3). In addition, _s(35/A366S) increased basal cAMP stimulation, consistent with the elevating effect of A366S on GDP release (21). In contrast, _s(35/E268A) exhibited the same dominant negative activity as _s(35) did (Fig. 1). Combining the two mutations that increased the dominant negative activity of _s(35), G226A and A366S, with the 35 substitutions resulted in a highly effective dominant negative _s construct that inhibited the cAMP accumulation response to 20 ng/ml hCG by 97% (S.E. = 0.2%, n = 3) (Fig. 1). No further increase in dominant negative activity resulted when the E268A substitution was added to _s(35/G226A/A366S) (Fig. 1).

The dominant negative _s mutants, _s(35), _s(G226A/E268A/A366S), and _s(35/G226A/A366S), decreased the effectiveness of signaling from the luteinizing hormone receptor to G_s both by increasing the EC₅₀ value of the cAMP response to hCG and by decreasing the magnitude of cAMP responses to hCG (Fig. 2 and Table I). Compared with _s(35) and _s(G226A/E268A/A366S), the effects of _s(35/G226A/A366S) on both of these parameters were greater. These incremental effects of combining the 35, G226A, and A366S mutations are consistent with their independent sites and mechanisms of action.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   s(35/G226A/A366S) causes greater inhibition of the cAMP response to luteinizing hormone receptor stimulation than s(G226A/E268A/A366S) and s(35) do. For each data point, 106 HEK-293 cells were transfected with 0.2 µg of plasmid encoding the luteinizing hormone receptor and 2 µg of vector (pcDNAI/Amp), or plasmid encoding s(G226A/E268A/A366S), s(35), or s(35/G226A/A366S). cAMP accumulation was measured in the absence or presence of the indicated amounts of hCG. All values represent the mean ± S.E. of three independent experiments performed in triplicate.

View this table: [in this window] [in a new window]   Table I EC50 values and percent inhibition of cAMP responses to hCG in HEK-293 cells expressing the luteinizing hormone receptor in the absence or presence of s mutants Values were determined in three experiments. Each value is the mean ± S.E.

Substitutions in the 35 Loop Rather Than in the 3 Helix Produce Dominant Negative Activity-- To investigate how altering the 35 loop region of _s produces dominant negative activity, the effects of smaller numbers of substitutions in this region were tested. Of the five _i2 homolog substitutions, two are located in the 3 helix (N271K and K274D) and three (R280K, T284D, and I285T) are in the 35 loop. Based on their location, only the loop residues are likely to interact directly with receptors (17). Separately testing the effects of substitutions in these two regions showed that the dominant negative effect is due predominantly to the substitutions in the 35 loop, rather than those in the 3 helix (Fig. 3). _s(R280K/T284D/I285T) was only slightly less effective as a dominant negative than _s(35) was, whereas _s(N271K/K274D) exhibited the same basal and receptor-stimulated cAMP accumulation as _s did. Therefore, the dominant negative phenotype of _s(35) appears to result from the alteration of a receptor contact site on _s.

View larger version (54K): [in this window] [in a new window]   Fig. 3.   Mutations in the 35 loop rather than the 3 helix of s produce dominant negative activity. For each data point, 106 HEK-293 cells were transfected with 0.2 µg of plasmid encoding the luteinizing hormone receptor and 2 µg of vector (pcDNAI/Amp) or plasmid encoding one of the indicated constructs. cAMP accumulation was measured in the absence (dark gray bars) or presence (light gray bars) of 20 ng/ml hCG. s(R280K/T284D/I285T), containing mutations in the 35 loop, exhibits dominant negative activity, whereas s(N271K/K274D), containing mutations in the 3 helix, exhibits normal basal and receptor-mediated activation. All values represent the mean ± S.E. of three independent experiments performed in triplicate.

Individual substitutions of each of the 35 loop residues decreased receptor-mediated activation of _s but did not produce dominant negative activity (Fig. 3). _s(R280K) exhibited very little basal or receptor-stimulated activity, whereas the activities of _s(T284D) and _s(I285T) were decreased relative to that of _s. The dominant negative activity produced by simultaneously substituting the three residues may result from additive defects in receptor interaction or may involve conformational changes in the loop due to interactions among the mutated residues.

The 35 Substitutions Compensate for the Instability Caused by the A366S Mutation-- A previously reported limitation of _s(G226A/E268A/A366S) is its instability (11), due to the A366S mutation, which increases the amount of time _s spends in the thermolabile nucleotide-free state (21). To determine whether the greater dominant negative activity of _s(35/G226A/A366S) compared with _s(G226A/E268A/A366S) is due in part to a stabilizing effect of the 35 substitutions, the expression levels of these _s constructs in membranes of transfected HEK-293 cells were compared (Fig. 4). The expression level of _s(35) was similar to that of _s, whereas that of _s(G226A/E268A/A366S) was much lower. The expression level of _s(35/G226A/A366S) was reduced somewhat relative to that of _s(35) and of _s but was much higher than that of _s(G226A/E268A/A366S). Thus, the 35 substitutions appear to counteract the destabilizing effect of A366S.

View larger version (62K): [in this window] [in a new window]   Fig. 4.   Expression levels of dominant negative s mutant constructs. HEK-293 cells (6.25 × 106) were transfected with 6 µg/106 cells of vector (pcDNAI/Amp) or plasmid encoding s(35), s(35/G226A/A366S), s(G226A/E268A/A366S), or s. s(G226A/E268A/A366S) is expressed at much lower levels than s is, whereas the expression level of s(35/G226A/A366S) is only slightly reduced relative to that of s. Similar results were obtained in two additional experiments.

_s(35/G226A/A366S) Inhibits Signaling of the Calcitonin Receptor to G_s and G_q-- To investigate how dominant negative _s activity affects the signaling pathways of G_s-coupled receptors that also interact with other G protein heterotrimers, signaling of the calcitonin receptor to G_s and G_q was monitored in the presence and absence of _s(35/G226A/A366S). _s(35/G226A/A366S) completely blocked _s-mediated cAMP accumulation in response to up to 0.48 nM sCT (Fig. 5A). Even at saturating amounts of sCT, this response was inhibited by 82.3% (S.E. = 0.3%, n = 3). _s(35/G226A/A366S) increased the EC₅₀ for stimulation of cAMP accumulation by sCT by a factor of 10 (Table II). As shown previously (9, 31), the calcitonin receptor coupled less efficiently to G_q than to G_s (Fig. 5 and Table II). In the presence of _s(35/G226A/A366S), _q-dependent inositol phosphate formation in response to all doses of sCT was blocked entirely (Fig. 5B).

View larger version (11K): [in this window] [in a new window]   Fig. 5.   s(35/G226A/A366S) inhibits signaling from the calcitonin receptor to Gs and Gq. 106 HEK-293 cells were co-transfected with 0.04 µg of plasmid encoding the rabbit C1a calcitonin receptor in pBKCMV (9) and 2 µg of vector (pcDNAI/Amp) (filled circles) or plasmid encoding s(35/G226A/A366S) (open circles). The average receptor number per cell was 282,000 (S.E. = 61,600, n = 3). A, inhibition of receptor-dependent cAMP accumulation by s(35/G226A/A366S). cAMP accumulation was measured in the absence or presence of the indicated amounts of sCT. B, inhibition of receptor-dependent inositol phosphate (IP) formation by s(35/G226A/A366S). Inositol phosphate formation was measured in the absence or presence of the indicated amounts of sCT. All values represent the mean ± S.D. from triplicate determinations in a single experiment. Two other experiments gave similar results.

View this table: [in this window] [in a new window]   Table II EC50 values of responses to sCT in HEK-293 cells expressing the calcitonin receptor in the absence or presence of s(35/G226A/A366S) EC50 values were determined in three experiments. Each value is the mean ± S.E. IP, inositol phosphate.

The ability of a dominant negative _s mutant to block signaling from the calcitonin receptor to both G_s and G_q leads to two conclusions regarding calcitonin receptor-G protein signaling. First, each calcitonin receptor has access to both _s and _q in transfected HEK-293 cells, suggesting that these  subunits share a common pool of receptors. Second, _s can compete with _q for binding to the calcitonin receptor.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Combining substitutions in three different regions of _s results in dominant negative _s activity that can inhibit signaling from G_s-coupled receptors by close to 100%. The substitutions affect distinct _s interactions as follows: increasing receptor affinity, decreasing receptor-mediated activation, and decreasing activation of adenylyl cyclase (35 substitutions), increasing affinity for (G226A), or decreasing affinity for GDP (A366S). Together, these mutations appear to stabilize the nucleotide-free -receptor complex. The incremental increases in dominant negative activity that result from combining these mutations are consistent with their independent sites and mechanisms of action. Although the nucleotide-free state, which is increased by the A366S mutation, is inherently unstable, _s(35/G226A/A366S) is expressed at a level close to that of wild-type _s. This is most likely because the increased receptor affinity caused by the 35 substitutions stabilizes the empty state, which has the highest receptor affinity (32). The 35 substitutions, on their own, do not affect the nucleotide handling properties of purified _s (17).

The ability of dominant negative _s activity to block signaling of the calcitonin receptor to multiple G protein pathways suggests that, at least in HEK-293 cells, distinct receptor-G protein complexes are not strictly compartmentalized into separate membrane domains. However, some mechanisms for compartmentalizing distinct receptor-G protein signaling pathways might not be detected using a dominant negative _s mutant. For instance, association of receptor subpopulations with distinct combinations could restrict potential  subunit interactions. Inactivation of specific G protein subunits using antisense (33-37) and ribozyme (38, 39) strategies has demonstrated a remarkable specificity of interaction between receptors, combinations, and effectors. In particular, in HEK-293 cells, ribozyme-mediated suppression of ₇ specifically reduced expression of ₁ and disrupted activation of G_s by -adrenergic but not prostaglandin E₁ receptors (38). Such specificity requirements might be overcome by a dominant negative _s mutant with increased affinity for both receptors and . In addition, although the ratio of G_s to its receptors in cells is generally ~100:1 (6), factors important for compartmentalization might become limiting when a dominant negative _s mutant is overexpressed. If this is so, then coexpressing potential compartmentalization factors with this _s mutant would be predicted to narrow the range of its inhibitory capacities.

The effect of dominant negative _s activity on other G protein pathways may depend on the cell and/or receptor type. For instance, cell-specific factors such as the caveolins, which have been reported to interact preferentially with particular G protein subunits (3), may restrict the accessibility of G proteins to receptors. HEK-293 cells do not have caveoli, although -adrenergic receptors and adenylyl cyclase V/VI localize to low buoyant density membrane domains in these cells (40). In addition, membrane fractionation studies have provided evidence for microdomains in the plasma membranes of neuroblastoma cells (41) and neutrophils (42) that have differences in their G protein content, and polarized epithelial cells differentially sort G protein  subunits (43) and G protein-coupled receptors (44). It will be of interest to use the dominant negative _s mutant described here to sort out the potential role of intracellular compartmentalization in regulating G protein signaling pathways in a wide range of cells and tissues.

The ability of a dominant negative _s mutant to inhibit signaling to G_q provides a molecular insight into receptor-G protein interactions in that it demonstrates that different types of G proteins can compete for binding to the calcitonin receptor. Previous studies of calcitonin receptor isoforms containing insertions or deletions identified distinct regions important for specific interactions but did not rule out mutually exclusive binding of different  subunits. The first intracellular loop (45-47) and an intact seventh transmembrane helix (9) appear to be important for coupling of this receptor to _q but not _s. The ability of different G proteins to compete for receptor binding despite these differences in specificity requirements indicates that either there is overlap in the receptor binding sites for different  subunits or that binding of one type of  subunit to a receptor sterically or allosterically blocks the association of a different one.

Competition between different G proteins for receptor binding raises the possibility that changes in the expression level of a particular G protein may affect other G protein signaling pathways as well. Alterations in G protein  subunit expression levels can take place on several time scales. Short term changes in the expression level of _s due to decreased stability of the activated state have been observed (48). More long term changes in the expression levels of _s (49) and _i2 (50) can occur during and play a role in development. In addition, pseudohypoparathyroidism is associated with decreased levels of functional _s (51).

The dominant negative _s mutant described here will have many applications to the investigation of how receptor-G protein signaling is regulated. In cell types for which there is strong evidence of subcellular compartmentalization, such as neurons and polarized epithelia, it may inhibit receptor subpopulations and be useful for determining which proteins coexist in the same membrane microdomains. In addition, it can be used to investigate the role of receptor-G protein interactions in the targeting of these proteins. For instance, if receptors are involved in the targeting of G protein subunits, receptor sequestration by a dominant negative  subunit may alter the localization patterns of wild-type subunits. The localization patterns of dominant negative  subunits as well as those of wild-type G protein subunits and receptors can be studied using fusions of these proteins to green fluorescent protein (52-55).

	ACKNOWLEDGEMENT

I thank Thomas Hynes for helpful discussions and critical reading of the text.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant GM50369 and American Heart Association Established Investigator Grant 9740043N.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 203-785-3202; Fax: 203-785-4951; E-mail: catherine.berlot@yale.edu.

Published, JBC Papers in Press, April 1, 2002, DOI 10.1074/jbc.M201330200

	ABBREVIATIONS

The abbreviations used are: hCG, human chorionic gonadotropin; sCT, salmon calcitonin.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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