©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
In Vivo Coupling of Insulin-like Growth Factor II/Mannose 6-Phosphate Receptor to Heteromeric G Proteins
DISTINCT ROLES OF CYTOPLASMIC DOMAINS AND SIGNAL SEQUESTRATION BY THE RECEPTOR (*)

(Received for publication, September 28, 1995)

Tsuneya Ikezu (1)(§)(¶) Takashi Okamoto(§) (2) Ugo Giambarella (2) Takashi Yokota (3) Ikuo Nishimoto (2)(**)

From the  (1)Shriners Hospitals for Crippled Children, Department of Anesthesia, Massachusetts General Hospital, Boston, Massachusetts 02114, the (2)Cardiovascular Research Center, Massachusetts General Hospital, Departments of Medicine, Harvard Medical School, Charlestown, Massachusetts 02129, and the (3)Department of Stem Cell Regulation, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We examined the signaling function of the IGF-II/mannose 6-phosphate receptor (IGF-IIR) by transfecting IGF-IIR cDNAs into COS cells, where adenylyl cyclase (AC) was inhibited by transfection of constitutively activated Galpha(i) cDNA (GalphaQ205L). In cells transfected with IGF-IIR cDNA, IGF-II decreased cAMP accumulation promoted by cholera toxin or forskolin. This effect of IGF-II was not observed in untransfected cells or in cells transfected with IGF-IIRs lacking Arg-Lys. Thus, IGF-IIR, through its cytoplasmic domain, mediates the G(i)-linked action of IGF-II in living cells. We also found that IGF-IIR truncated with C-terminal 28 residues after Ser caused Gbeta-dominant response of AC in response to IGF-II by activating G(i). Comparison with the Galpha(i)-dominant response of AC by intact IGF-IIR suggests that the C-terminal 28-residue region inactivates Gbeta. This study not only provides further evidence that IGF-IIR has IGF-II-dependent signaling function to interact with heteromeric G proteins with distinct roles by different cytoplasmic domains, it also suggests that IGF-IIR can separate and sequestrate the Galpha and Gbeta signals following G(i) activation.


INTRODUCTION

Insulin-like growth factor II (IGF-II) (^1)promotes growth, mainly in fetal development. In cultured cells, it exerts mitogenic and metabolic stimulation by binding to cell surface receptors. IGF-IIR is a high-affinity receptor for IGF-II(1, 2, 3) . It is also a receptor for M6P(3) . However, these two distinct ligands bind to different sites in IGF-IIR, which has been indicated by competition experiments and by the fact that only mammalian IGF-IIR can bind IGF-II(4) . For several reasons (for review, see (4) ), it remains unclear whether the IGF-IIR executes signaling functions in response to IGF-II. Nonetheless, there are multiple lines of independent evidence that IGF-IIR has signaling function activated by IGF-II. In multiple cultured-cell systems, IGF-II evokes cellular responses, most likely through IGF-IIR(3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) . We and another group independently showed that IGF-II stimulation of IGF-IIR promotes calcium influx through G(i), a member of the heteromeric G protein family, in Balb/c3T3 or CHO cells(12, 13, 14, 15) . In reconstituted vesicles, purified IGF-IIR directly couples to G(i) in response to IGF-II(16, 17, 18) , and human IGF-IIR has a cytoplasmic 14-residue region at Arg-Lys, which can directly activate Galpha(i)(18, 19) . In cell-free systems, this region most likely functions as the effector domain of IGF-IIR for G(i) coupling(18, 19, 20) . Although failure of IGF-IIR coupling to G proteins in cell-free systems was once reported(21) , a subsequent paper(15) , with two of the same authors, suggested the G(i) coupling function of IGF-IIR, based on the observation that IGF-II stimulates Ca influx via a pertussis toxin-sensitive G protein in a manner resistant to tyrosine kinase inhibitors.

Intensive studies of the molecular signaling function of IGF-IIR have so far been conducted only on cell-free experimental systems, which have serious limitations. The present study was conducted to establish a more physiological system, where one can investigate the signaling functions of IGF-IIR. Here we report that IGF-II links recombinant IGF-IIR to the G(i)/AC system in living cells. Furthermore, to the extreme C terminus of IGF-IIR, we assigned a novel function of inactivating Gbeta, which is another component of heteromeric G proteins. This study not only offers further evidence for the interaction of IGF-IIR with heteromeric G proteins, it provides a novel insight into the differential regulation of G protein subunit signals by receptors.


EXPERIMENTAL PROCEDURES

Galpha(t) and gip2 (GalphaQ205L) cDNAs were provided by Dr. H. R. Bourne. Wild-type Galpha cDNA, human IGF-IIR cDNA, Delta2410-2423 cDNA, and the construction method of IGF-IIR mutants were described previously(20, 22) . Oligonucleotide-directed mutagenesis was done to construct DeltaCT41 and DeltaCT28 according to the Kunkel method(23) . Oligonucleotides used were GAGCGTGAGGACGATTGATGAAGGGTGGGGCTGGTC for DeltaCT41, and GCGAGGAAAGGGAAGTGATGATCCAGCTCTGCACAG for DeltaCT28.

COS cells were grown in DMEM plus 10% calf serum and streptomycin/penicillin. For stable expression of Galpha(t), COS cells were transfected by the calcium phosphate method using 10 µg of Galpha(t) cDNA and 0.3 µg of pBabe/Puro, a puromycin resistance gene. Cells were then selected with 3 µg/ml puromycin and tested for immunoblot analysis with AS/7. The COS cell line used here expresses Galpha(t) at an approximately half the level of endogenous Galpha(i).

Plasmids were transfected by the lipofection method as described(22) . Intracellular accumulation of cAMP was measured as described(24) . A day before transfection, 5 times 10^4 cells were seeded on a 12-well plate. Unless otherwise specified, cDNAs encoding IGF-IIRs or Galpha/gip2 (0.5 µg/ml each) were transfected with 1 µl/ml LipofectAMINE (Life Technologies, Inc.) and incubated for 24 h in a serum-free culture. After washing cells with fresh media, cells were labeled with 3 µCi of [^3H]adenine for another 23 h 30 min. It should be emphasized that cells were then washed rigorously with solution containing M6P, as follows. DMEM-Hepes (DMEM containing 25 mM Hepes/NaOH, pH 7.4) containing 10 mM M6P was added to cells after discarding media. Cells were then incubated for 15 min at room temperature and washed four times with DMEM-Hepes. These procedures, which dissociate M6P and M6P-containing proteins from IGF-IIR, ensured reproducibility for inhibition of AC by IGF-IIR stimulation. This was reasonable because M6P binding to IGF-IIR impaired the action of IGF-II to inhibit AC in cells transfected with IGF-IIR cDNA (see ``Results''). Cells were then treated with 2.5 µg/ml CTX (Calbiochem) and 1 mM isobutylmethylxanthine with or without IGF-II (or IGF-I) in DMEM-Hepes at 37 °C for 30 min. Reactions were terminated by aspiration and the immediate addition of 5% ice-cold trichloroacetic acid (1 ml/well). Acid-soluble nucleotides were separated on two-step ion-exchange columns as described(24) , and specific accumulation of cAMP is expressed as (cAMP/ADP + ATP) times 10^3.

For binding assay of IGF-IIR, cells (3 times 10^6/dish) were transfected with 10 µg of IGF-IIR cDNAs and 20 µl of LipofectAMINE in 5 ml of DMEM plus streptomycin/penicillin. Twenty-four hours after transfection, the medium was renewed to DMEM plus 10% calf serum and streptomycin/penicillin. By scraping cells 48 h after transfection, membranes were prepared and IGF-II binding assay was performed as described(20) . Specific binding was calculated by subtracting nonspecific binding, the binding in the presence of 100 nM IGF-II. All other materials were obtained from commercial sources. Data were analyzed with Student's t test.


RESULTS AND DISCUSSION

We initially examined whether our COS cells were appropriate to see the effects of Galpha(i). Indeed, COS cells have not frequently been used to examine the effects of G(i) or G(i)-coupled receptors, although Bell and co-workers (25) have described the G(i)-coupled effect of somatostatin receptors using COS cells. For this reason, we tested the effect of transfection of wild-type Galpha or constitutively activated Galpha mutant gip2 cDNA on AC activity. As shown in Fig. 1A, transfection of gip2 resulted in dose-dependent inhibition of CTX-stimulated cAMP accumulation, whereas that of wild-type Galpha had no effect. Therefore, our COS cells seemed to be suitable for examining the G(i)-coupling function of receptors with transient transfection of cDNAs.


Figure 1: Effects of activated Galpha and intact IGF-IIR on AC activity in COS cells. A, dose effect of transfection of either gip2 cDNA or wild-type Galpha cDNA on CTX-stimulated AC activity in COS cells. AC activity is indicated as a percentage of CTX-stimulated activity in mock-transfected COS cells, which was 27.2 ± 2.9 (cAMP/(ADP+ATP) times 10^3). Each value represents the mean ± S.E. of single determinants done with four independent transfections. *, p < 0.05;**, p < 0.01 versus no cDNA. B, specific IGF-II binding to the membranes of transfected COS cells. COS cells were transfected with each IGF-IIR or mutant cDNA. Forty-eight h after transfection, membranes were prepared and specific IGF-II binding was measured. The results are representative of four independent transfections, which yielded similar results. C, dose effect of IGF-II on CTX-stimulated AC activity in IGF-IIR-transfected COS cells. COS cells were transfected with 0.5 µg/ml IGF-IIR cDNA. Cells were stimulated by 2.5 µg/ml CTX in the presence of various concentrations of IGF-II. AC activity was assessed by measuring (cAMP/(ADP + ATP)) times 10^3. Each value represents the mean ± S.E. of single determinants done with four independent transfections. Thus, the effect of IGF-II was highly reproducible across transfections. *, p < 0.05;**, p < 0.01 versus no IGF-II. D, effect of IGF-II on forskolin-stimulated AC activity in COS cells transfected with IGF-IIR. After transfection of IGF-IIR cDNA, cells were treated with increasing concentrations of forskolin with or without 10 nM IGF-II. AC activity is indicated as n-fold of basal activity in these cells, which was 0.345 ± 0.10. The S.E. of AC activity in the presence of 1 µM forskolin stimulation without IGF-II was 2.4. Each value represents the mean ± S.E. of single determinants done with four independent transfections.**, p < 0.05 versus no IGF-II



Intact IGF-IIR cDNA was transfected into these COS cells, which were treated with IGF-II before cell lysis. Parental COS cells expressed 3.6 fmol/µg of endogenous IGF-II binding sites having the K of 0.90 nM. With cDNA transfection, these cells expressed recombinant IGF-IIRs with comparable affinities by severalfold of the endogenous binding level (Fig. 1B, B(max) and K were 7.3 fmol/µg and 1.3 nM in intact IGF-IIR transfection, 9.5 fmol/µg and 1.4 nM in DeltaCT41 transfection, 7.1 fmol/µg and 0.85 nM in DeltaCT28 transfection, and 6.0 fmol/µg and 0.88 nM in Delta2410-2423 transfection, respectively). The endogenous IGF-II binding site appears to be virtually IGF-IR, because only a 110-kDa protein was cross-linked with radioactive IGF-II in an IGF-II-inhibitable manner in parental COS cell membranes under the same condition as in the IGF-II binding assay (data not shown). This assessment is not only consistent with the report of Steele-Perkins et al.(26) that IGF-IR exhibits considerably high affinity for IGF-II, but is also strongly supported by the report of Oshima et al.(27) showing that endogenous IGF-IIR is scarce in COS cells.

In the cells transfected with either gene, 2.5 µg/ml CTX constantly increased AC activity by 5-fold over the basal level. In cells transfected with intact IGF-IIR cDNA, IGF-II significantly impaired the CTX-stimulated AC activity in a dose-dependent manner (Fig. 1C). IGF-II also inhibited forskolin-stimulated AC activity (Fig. 1D). It is underscored that no inhibition of AC was observed by IGF-II without transfection of IGF-IIR cDNA (Fig. 2). Thus, the effect of IGF-II was attributable to the recombinant IGF-IIR. In accord with this idea, 10 nM IGF-I did not reproduce the effect of IGF-II (Fig. 2). Consistent with our previous study(12, 13, 16, 17, 20) , this inhibitory effect of IGF-II was abolished by PTX and by 10 mM M6P (Table 1). These data indicate that IGF-II triggers the signaling function of IGF-IIR and couples it to the Galpha(i)/AC system in living cells.


Figure 2: Effect of IGF-IIR and cytoplasmic mutants on AC activity in COS cells. COS cells were transfected with recombinant IGF-IIR cDNAs or pECE vector (each 0.5 µg/ml). Cells were then stimulated by 2.5 µg/ml CTX in the presence or absence of 10 nM IGF-II, and AC activity was measured. As a control, the effect of 10 nM IGF-I was examined in the IGF-IIR-transfected COS cells. Each value represents the mean ± S.E. of single determinants done with three independent transfections. AC activity is indicated as a percentage of CTX-stimulated activity in mock-transfected COS cells, which was similar to that in the left panel C.***, p < 0.005. n. s., not significant. Inset, illustration of IGF-IIR mutants.





In cell-free systems, the Arg-Lys region of IGF-IIR has been implicated in its G(i) coupling function(18, 19, 20) . To examine whether this is the case in living cells, we constructed mutant IGF-IIRs lacking the C-terminal 41 residues after Arg (DeltaCT41) or the 28 residues after Ser (DeltaCT28) and lacking Arg-Lys (Delta2410-2423). Despite remarkable expression of DeltaCT41 (Fig. 1B), IGF-II failed to inhibit CTX-stimulated AC activity in cells transfected with this mutant (Fig. 2), indicating an essential role of the C-terminal 41 residues for AC suppression. We unexpectedly found a novel AC-linked function of DeltaCT28. In DeltaCT28-transfected COS cells, CTX augmented AC activity to the same level as in COS cells transfected with other IGF-IIRs; however, only in the DeltaCT28-transfected cells, did IGF-II further potentiate CTX-stimulated AC activity (Fig. 2). This effect of IGF-II depended on the amount of DeltaCT28 cDNA used for transfection (Fig. 3A). In the same DeltaCT28-transfected cells, IGF-II did not affect AC activity without CTX (not shown). These suggest that AC potentiation by DeltaCT28 is mediated by the Gbeta subunit of heteromeric G proteins(28, 29) .


Figure 3: IGF-II-dependent augmentation of AC stimulation by IGF-IIRDeltaCT28. A, effect of DeltaCT28 cDNA on CTX-stimulated AC activity in COS cells. COS cells were transfected with increasing doses of DeltaCT28 cDNA and stimulated by 2.5 µg/ml CTX with or without 10 nM IGF-II. Basal AC activity in mock-transfected COS cells was 2.0 ± 0.1. Each value represents the mean ± S.E. of single determinants done with four independent transfections. *, p < 0.02;**, p < 0.05 versus no IGF-II. B, effect of DeltaCT28 on CTX-induced AC stimulation in COS cells overexpressing Galpha(t) (COS/Galpha(t)). COS/Galpha(t) cells were transfected with DeltaCT28 cDNA (or vector), and stimulated by 2.5 µg/ml CTX with or without 10 nM IGF-II. AC activity is indicated as a percentage of CTX-stimulated activity in mock-transfected COS/Galpha(t) cells, which was 22.7 ± 2.6. Each value represents the mean ± S.E. of four independent experiments. Note that IGF-II augmented CTX-stimulated AC activity by 200% in parental COS cells that received DeltaCT28 transfection under the same condition. C, effect of PTX on IGF-II/DeltaCT28-induced augmentation of CTX-stimulated AC activity. COS cells were transfected with DeltaCT28 for 24 h and treated with 10 ng/ml PTX for another 24 h. During the last 30 min, cells were stimulated by 2.5 µg/ml CTX with or without 10 nM IGF-II. AC activity is indicated as a percentage of CTX-stimulated activity in DeltaCT28-transfected COS cells. Each value represents the mean ± S.E. of four independent experiments. D, effect of IGF-IIRDelta2410-2423 on AC activity in COS cells. COS cells were transfected with Delta2410-2423 cDNA and then stimulated by 2.5 µg/ml CTX in the presence or absence of 10 nM IGF-II, and AC activity was measured. Each value represents the mean ± S.E. of four independent experiments. AC activity is indicated as percentage of the CTX-stimulated activity in Delta2410-2423-transfected COS cells, which was 20.7 ± 2.8.



To confirm the Gbeta mediation, we examined the effect of Galpha(t) on the function of DeltaCT28 (Fig. 3B). In COS cells overexpressing Galpha(t) (COS/Galpha(t)), CTX stimulated AC activity with a similar fold of the basal activity to that observed in parental COS cells and DeltaCT28-transfected COS cells. In COS/Galpha(t) cells transfected with DeltaCT28, IGF-II hardly potentiated the CTX-stimulated AC activity, indicating that Galpha(t) impaired this function of DeltaCT28. The reason why DeltaCT28 could not significantly inhibit AC activity in COS/Galpha(t) cells was likely that Galpha(t) expression was not sufficient to totally absorb the Gbeta released by DeltaCT28 stimulation. Since Galpha(t) acts as a specific inhibitor of Gbeta without affecting AC(29) , these data confirm that Gbeta mediates the IGF-II-triggered potentiation of AC by DeltaCT28.

As high concentrations of Gbeta are required to enhance AC activity, the source of Gbeta should be G(i) in non-neuronal cells(30) . It was thus likely in our COS cells, that DeltaCT28 releases Gbeta by activating G(i). To confirm the source of Gbeta, we examined the effect of PTX on this function of DeltaCT28. As in Fig. 3C, 24-h treatment of 10 ng/ml PTX blocked the stimulatory effect of 10 nM IGF-II in COS cells transfected with DeltaCT28, while the CTX response was not changed by PTX. These data indicate that the action of DeltaCT28 on Gbeta is through G(i), again suggesting that the Arg-Lys region is interactive with G(i).

We confirmed the inability of Delta2410-2423 to affect AC (Fig. 3D). In COS cells transfected with Delta2410-2423, IGF-II could neither inhibit nor augment AC activity, despite the expression of this mutant comparable to that of intact IGF-IIR (Fig. 1B). Delta2410-2423 is mutant IGF-IIR that lacks Arg-Lys but retains the extreme C-terminal 28 residues that DeltaCT28 lacks. Therefore, this finally demonstrates that the domain that is essential for the interaction with G(i) is not the extreme C terminus but the Arg-Lys region.

We have herein established a whole-cell system in which IGF-II-triggered signaling function of IGF-IIR can be examined. Using this system, multiple lines of evidence show that recombinant human IGF-IIR activates Galpha(i) and suppresses AC in response to IGF-II. IGF-IIR transfection was required to observe the effect of IGF-II on AC, consistent with the scarceness of endogenous IGF-IIR in COS cells. IGF-I could not reproduce the effect of IGF-II. In addition, M6P treatment of transfected COS cells blocked the effect of IGF-II, reproducing our in vitro data(16) . It is thus emphasized that insufficient removal of lysosomal enzymes from IGF-IIR precludes this receptor from responding to IGF-II. Furthermore, the Arg-Lys region is shown here to be essential for AC suppression by IGF-IIR, as predicted by our in vitro study(18, 19, 20) .

In this study, an IGF-IIR mutant has pointed to a novel function of the receptor C terminus. DeltaCT28 enhanced cAMP production in response to IGF-II. Multiple lines of evidence indicate that this response was mediated by Gbeta, the source of which was the activated G(i). In contrast, intact IGF-IIR activated G(i) and mainly generated the signal of Galpha(i) (inhibition of AC) in response to the same stimulation. These indicate that the C-terminal Ser-Ile region of IGF-IIR can inactivate Gbeta. This inactivation suggests direct or indirect interaction of this C-terminal region with Gbeta. In further support of this idea, the Ser-Ile region is homologous to a part of the PH domains (31) of multiple proteins including beta-adrenergic receptor kinase (Fig. 4), which are proven to bind Gbeta(32) . It has also been shown that the isolated PH domain of beta-adrenergic receptor kinase inactivates the action of Gbeta(33) . These findings suggest that this region of IGF-IIR inactivates Gbeta through interaction. Because of technical difficulty, we were not able to examine the effects of the isolated regional peptides on AC augmentation by DeltaCT28 observed in this whole-cell system.


Figure 4: The C terminus of human IGF-IIR is homologous to multiple PH domains. Ile is the C-terminal end of human IGF-IIR. Identical residues are in black, and similar residues are shaded. The sequences of the PH domains all correspond to their sixth subdomains.



Among known AC subtypes, no single AC that responds to both Galpha(i) (inhibitory) and Gbeta (stimulatory) has been specified. However, among AC types I-VI, our COS cells express type VI, which is inhibited by Galpha(i), and type IV, which is stimulated by Gbeta (data not shown). It is thus reasonable to assume that the whole response of AC to G(i) in intact COS cells is the sum of the respective effects of Galpha(i) and Gbeta on these AC subtypes, thus allowing the total AC activity to respond to both G-protein subunits.

In summary, this study shows that IGF-IIR, in living cells, activates G(i) and affects AC through differential actions of multiple cytoplasmic domains of its own. In our cells, it activates G(i) through Arg-Lys and inactivates Gbeta through Ser-Ile, resulting in the predominant action of Galpha(i). Therefore, the distinct roles played by multiple domains of IGF-IIR separate and sequestrate the Galpha and Gbeta signals following G(i) activation. This is potentially a very interesting mechanism that allows a receptor to differentially activate Galpha and Gbeta and selectively turn on each subunit-specific pathway. With this novel mechanism, there is no longer necessity that a receptor must always turn on both subunit pathways by activating one heteromeric G protein complex. It is thus important to investigate whether a similar mechanism is possessed by other receptors.

It is also conceivable that this novel property of IGF-IIR may contribute to its unique signaling function in vivo. Multiple effects of G(i) depend on G(i)-released Gbeta(34) . Thus, IGF-IIR-induced G(i) activation may lack some of the G(i) outputs induced by conventional receptors. It is also conceivable that the Gbeta inactivating effect of the C terminus of this receptor may be affected by the amount of free Gbeta inside the cell. There may be an intracellular free Gbeta pool with different sizes in different cells(35) . Excess Gbeta may thus occupy the C terminus and attenuate its inhibitory effect. In accord with this idea, IGF-II binding to IGF-IIR can potentiate AC stimulation in human fibroblasts (36) but not in COS cells (this report) and can stimulate PI turnover in renal cells (5) but not in Balb/c3T3 (12) or CHO cells (15) . Alternatively, the Gbeta-linked function of IGF-IIR might be involved in its trafficking function as an M6P receptor, as one of the established functions of Gbeta is translocation of target proteins (37) . This is, however, less likely, because the residues essential for IGF-IIR trafficking have been mainly localized near the N terminus of the cytoplasmic domain, particularly before Arg(38) . This possibility is further lowered by the fact that the cation-dependent M6P receptor, another trafficking receptor for M6P, has no cytoplasmic regions homologous to the PH-like domain in the extreme C terminus of IGF-IIR. In conclusion, this study demonstrates the coupling of IGF-IIR with heteromeric G proteins in native cell environments. While calcium influx is one of its most likely outputs(12, 13, 14, 15) , it is important to determine which cellular function is executed by the demonstrated IGF-IIR interaction with the G proteins.


FOOTNOTES

*
This work was supported in part by Bristol-Myers Squibb. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
The first two authors contributed equally to this work.

Recipient of a fellowship from JSPS.

**
To whom correspondence should be addressed. Tel.: 617-726-3902; Fax: 617-726-5806; nishimoto@helix.mgh.harvard.edu.

(^1)
The abbreviations used are: IGF-I and -II, insulin-like growth factors I and II, respectively; IGF-IR and IGF-IIR, the receptors for IGF-I and IGF-II, respectively; M6P, mannose 6-phosphate; AC, adenylyl cyclase; DMEM, Dulbecco's modified Eagle's medium; DeltaCT41 or DeltaCT28, mutant IGF-IIR lacking the C-terminal 41 residues after Arg or the 28 residues after Ser, respectively; Delta2410-2423, mutant IGF-IIR lacking Arg-Lys; Galpha, alpha subunit of G; Galpha(t), alpha subunit of transducin; CTX, cholera toxin; PTX, pertussis toxin; PH, pleckstrin homology.


ACKNOWLEDGEMENTS

We are grateful to M. C. Fishman, T. B. Kinane, and D. A. Ausiello for their critical reading of this manuscript; H. R. Bourne for Galpha cDNAs and critical reading of the prior version of this manuscript; and D. Wylie for expert technical assistance.


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