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CELLULAR AND MOLECULAR

Mutational Uncoupling of _1A-Adrenergic Receptors from G Proteins Also Uncouples Mitogenic and Transcriptional Responses in PC12 Cells

Department of Pharmacology, Emory University Medical School, Atlanta, Georgia

Received February 14, 2003; accepted April 16, 2003.

	Abstract

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Activation of human _1A-adrenergic receptors in PC12 cells causes many second messenger, mitogenic, and transcriptional responses. We examined the role of G protein activation in these responses by uncoupling the receptor through deletion of the first three amino acids from the third intracellular loop (^208–210). Expression levels of retrovirus-transfected wild-type and ^208–210 _1A-adrenergic receptors in PC12 cells were similar and showed identical binding affinities for antagonists. However, the potency of the agonist norepinephrine was increased 9-fold by the ^208–210 mutation. In PC12 cells expressing the ^208–210 construct, calcium and inositol phosphate responses to norepinephrine were essentially abolished. The strong activation of mitogen-activated protein kinase pathways seen upon stimulation of wild-type _1A-adrenergic receptors in PC12 cells was abolished by the ^208–210 mutation, as was activation of the tyrosine kinase Pyk2. Norepinephrine also activates several transcriptional reporters through _1A-adrenergic receptors in PC12 cells, including reporters for activator protein 1, serum response element, cAMP response element, nuclear factor-B, nuclear factor of activated T cells, -interferon-activated sequence, and signal transducer and activator of transcription. All these transcriptional responses were abolished by the ^208–210 mutation. Overexpression of G16 did not rescue any of these responses. These data suggest that known second messenger, mitogenic, and transcriptional effects of _1A-adrenergic receptors in PC12 cells all require G protein activation.

We studied the signaling events activated by human ₁-ARs stably expressed in PC12 cells. Like other GPCRs, ₁-ARs activate mitogenic responses in many cells and play important roles in regulating growth and proliferation (Zhong and Minneman, 1999b). In rat PC12 cells stably transfected with _1A-ARs, norepinephrine (NE) activates a variety of mitogen-activated protein kinase (MAPK) pathways and causes the cells to differentiate into a neuronal like phenotype similar to that caused by exposure to nerve growth factor (NGF) (Williams et al., 1998; Zhong and Minneman, 1999c). These responses are associated with activation of a variety of tyrosine kinases, particularly Pyk2 and Src (Zhong and Minneman, 1999a). In addition, NE activates a series of transcriptional reporters in _1A-transfected PC12 cells, including reporters containing consensus binding sequences for AP1, SRE, CRE, NF-B, NFAT, GAS, and Stat (Minneman et al., 2000; Zhong et al., 2000). Because the use of inhibitors suggests that many of the mitogenic, tyrosine kinase, and transcriptional responses are independent of the normal Gq-mediated inositol phosphate and calcium second messenger responses produced by receptor activation (Berts et al., 1999), we wondered whether some of these responses might be independent of G protein activation.

Several mutations have been reported to disrupt coupling of hamster _1B-ARs to Gq family members. Wu et al. (1995) reported that deletion of portions of the third intracellular loop blocked coupling of the hamster _1B-AR to Gq family members. The shortest effective deletion was the loss of the first three amino acids (217–219) from the N terminus of the third intracellular loop. In addition, Wang et al. (1997) found that mutation of the conserved tyrosine 348 to alanine in the conserved NPXXY motif of the seventh transmembrane domain of the hamster _1B-AR resulted in a receptor that was unable to activate second messenger pathways.

We have now made analogous mutations in the human _1A-AR, which we have previously shown to cause the strongest mitogenic and transcriptional responses in PC12 cells. We report that deletion of the N-terminal three amino acids from the third intracellular loop results in a receptor with an uncoupled phenotype, and use this receptor to determine whether any of the responses we have observed are independent of G protein activation.

	Materials and Methods

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Cell Culture. PC12 cells were propagated in 75-cm² flasks at 37°C in a humidified 5% CO₂ incubator in Dulbecco's modified Eagle's medium containing 4.5 g/l glucose, 1.4% glutamine, 20 mM HEPES, 100 µg/l streptomycin, 10 U/ml penicillin, 10% donor horse serum, and 5% fetal bovine serum (Williams et al., 1998). A PC12 cell subclone stably expressing wild-type human _1A-ARs under control of an isopropyl -D-thiogalactoside-inducible promoter (Williams et al., 1998) (_1A-28) was used as a control. HEK293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 µg/ml streptomycin, and 100 U/ml penicillin at 37°C in a humidified atmosphere with 5% CO₂.

Site-Directed Mutagenesis. The desired mutations were produced by polymerase chain reaction with the QuikChange kit (Stratagene, La Jolla, CA) and confirmed by sequencing.

Retroviral Plasmids and Transfection. All wild-type and mutated constructs were subcloned into the plasmid pTJM9 (Boss et al., 1998) for retrovirus production. Phoenix-producer cells (American Type Culture Collection, Manassas, VA) were purchased by permission of Dr. Gary Nolan (Stanford University, Palo Alto, CA) for transient production of nonreplicating recombinant retrovirus. Infectious retroviral supernatants were generated by a helper virus-free protocol and PC12 cells infected as described elsewhere (Abbott et al., 2000). Infected cells were selected by exposure to 0.5 mg/ml geneticin for 2 weeks. In some experiments, human G16 (Amatruda et al., 1991) was subcloned into a plasmid pTJ66 (Murphy et al., 2002) containing a zeocin-resistance marker. Retrovirus was produced and PC12 cells stably expressing the ^208–210 _1A-AR were infected. Transfected cells were selected by exposure to 1 mg/ml zeocin for 2 weeks and G16 expression confirmed by Western blotting.

Radioligand Binding. Cells were homogenized with a Polytron, membranes collected by centrifugation, and receptor density determined by saturation analysis of specific binding of the ₁-AR antagonist radioligand ¹²⁵I-BE 2254 (20–800 pM) (Theroux et al., 1996). For competition curves, 50 pM radioligand was used. Nonspecific binding was defined as binding in the presence of 10 µM phentolamine.

Inositol Phosphate Formation. Accumulation of [³H]inositol phosphates was determined in 35-mm dishes. Cells were labeled with [³H]myo-inositol (2 µCi/plate) for 1 to 2 days and production of total [³H]inositol phosphates in the presence of 10 mM LiCl determined as described previously (Esbenshade et al., 1993).

Intracellular Ca²⁺ Concentration Determinations. Intracellular Ca²⁺ concentration transients were determined using fura-2 as described previously (Esbenshade et al., 1993).

Immunoblotting. Confluent cells were serum-starved for 2 h before use, and drug treatments were generally carried out for 15 min at 37°C. After stimulation, cells were lysed, centrifuged, proteins resolved by SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose. Phosphorylation of ERKs and JNKs was detected by immunoblotting using antibodies to dually phosphorylated active forms of the enzymes (Williams et al., 1998). Protein tyrosine phosphorylation was detected by immunoprecipitation with an anti-Pyk2 antibody and immunoblotted using an anti-phosphotyrosine antibody (Zhong and Minneman, 1999a). Human 16 immunoreactivity was detected with an antibody generously provided by Dr. John Hepler (Emory University, Atlanta, GA).

Luciferase Reporters. PC12 cells were further transfected with retrovirus coding for luciferase reporters containing consensus sequences for various transcription factors (Abbott et al., 2000). Luciferase activity was determined as described previously (Minneman et al., 2000).

Coimmunoprecipitation of G_q(EE). Coimmunoprecipitation of G_q(EE) (Guthrie Research Institute, Sayre, PA; courtesy of Dr. John Hepler) with FLAG-tagged _1A- or ^208–210 _1A-AR was performed after cotransfection into HEK293 cells (Chen et al., 2000). Cells were harvested in 1 ml of Tris buffer (50 mM Tris, 150 mM NaCl, 5 mM MgCl₂, 1 mM EGTA, 1 mM EDTA, and 1 mM dithiothreitol, pH 7.4) containing protease inhibitors (1 mM benzamidine, 2 µg/ml pepstatine, 2 µg/ml aprotinin, 2 µg/ml leupeptin, and 200 µM phenylmethylsulfonyl fluoride). Membranes were prepared by sonication on ice for three 10-s intervals, and treated with (–)-norepinephrine (0.1 mM) at room temperature for 30 min and throughout the immunoprecipitation. Membranes were solubilized with 1% digitonin and sonicated for 10 s before incubation at 4°C for 3 h. The solubilized fraction was centrifuged at 100,000g for 30 min at 4°C and the supernatant mixed with 100 µl of anti-FLAG M2 affinity gel (Sigma-Aldrich, St. Louis, MO) in 0.1% digitonin and incubated overnight at 4°C. After three washes with Tris buffer containing 0.1% digitonin, the affinity gel was resuspended in Laemmli SDS sample buffer and incubated at room temperature for 1 h before electrophoresis on a 4 to 12% Tris-glycine gel (Novex, San Diego, CA). After transfer to nitrocellulose, samples were immunoblotted for G_q using a monoclonal antibody against the EE epitope (1:1000; Covance, Berkeley, CA) and horseradish peroxidase-conjugated goat anti-mouse IgG antibody (1:3000; Promega, Madison, WI). Bands were visualized by an enhanced chemiluminescence system (PerkinElmer Life Sciences, Boston, MA).

	Results

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Expression of Mutant Receptors. Two separate mutations analogous to those previously reported to uncouple hamster _1B-ARs were made in the human _1A-AR. These include deletion of the first three amino acids at the N terminus of the third intracellular loop (Wu et al., 1995) (^208–210) and mutation of the tyrosine in the conserved NPXXY motif in the seventh transmembrane domain to alanine (Y326A) (Wang et al., 1997). Preliminary determinations of the expression of these constructs were obtained by transient transfection into HEK293 cells. We found good expression of the ^208–210 construct using radioligand binding, but no detectable expression of the Y326A construct (data not shown).

Pharmacological Characterization. The ^208–210 construct was expressed in PC12 cells using retrovirus (Minneman et al., 2000), and stably transfected cells were selected with geneticin. We used a previously described PC12 cell line stably expressing wild-type human _1A-ARs (_1A-28) (Berts et al., 1999) for comparison. Figure 1 shows that these two cell lines exhibited a similar density of binding sites for the antagonist radioligand ¹²⁵I-BE 2254 (B_max: 313 ± 6 fmol/mg of protein wild type; 290 ± 13 fmol/mg of protein ^208–210) with similar affinities (K_D: 30 ± 12 pM wild type; 52 ± 8 pM ^208–210). Competition experiments showed that the two constructs showed identical affinities for the ₁-selective antagonist prazosin, the _1A-selective antagonist (+)-niguldipine, and the _1D-selective antagonist BMY7378 (Fig. 2). However, the ^208–210 construct showed a significant 9-fold higher affinity for the agonist NE than did the wild-type receptor (Fig. 2). This increased agonist affinity has been observed previously in uncoupled mutations of other GPCRs (Strader et al., 1987; Wang et al., 1997).

View larger version (25K): [in this window] [in a new window]   Fig. 1. Saturation binding of 125I-BE 2254 (125I-BE) to wild-type (1A-28) or mutant (208–210) 1A-ARs in stably transfected PC12 cells. Cells were transfected with wild-type (1A-28) or mutant (208–210) 1A-ARs using retrovirus, selected with geneticin, and propagated as described under Materials and Methods. After harvesting, membranes were prepared and saturation binding of 125I-BE determined as described. The results represent the mean ± S.E.M. of results from four experiments performed in duplicate.

View larger version (30K): [in this window] [in a new window]   Fig. 2. Comparison of antagonist and agonist affinities at wild type (1A-28) or mutant (208–210) 1A-ARs in stably transfected PC12 cells. Cells stably expressing wild-type (1A-28) or mutant (208–210) 1A-ARs were harvested and membranes prepared as described. Competition for 125I-BE 2254 (125I-BE binding) (50 pM) was determined for the indicated drugs as described in the text. The results represent the mean ± S.E.M. of results from three to four experiments performed in duplicate. The –log KI values for wild-type and mutant receptors were as follows: prazosin 8.6 ± 0.05 M, 8.6 ± 0.05 M; (+)-niguldipine 8.9 ± 0.11 M, 8.9 ± 0.10 M; BMY 7378 7.1 ± 0.03 M, 7.3 ± 0.08 M; and NE 5.2 ± 0.04 M, 6.1 ± 0.04 M.

Inositol Phosphate and Calcium Responses. The effect of NE on inositol phosphate and calcium levels was examined in PC12 cells stably expressing wild-type or ^208–210 _1A-ARs. Figure 3 shows that NE increased [³H]inositol phosphate formation by 17-fold in _1A-28 PC12 cells. This response was almost completely eliminated in PC12 cells expressing the ^208–210 construct, although NE did cause a statistically significant 0.5-fold increase over basal inositol phosphate formation in ^208–210 expressing cells that was not observed in untransfected cells. UTP caused a similar doubling in [³H]inositol phosphate formation in both cell lines that was highly statistically significant. Figure 4 shows that NE caused no detectable increase in intracellular calcium in cells expressing the ^208–210 construct, although NE caused about a 3-fold increase in wild-type _1A-28 PC12 cells. The purinergic agonist UTP, which acts through endogenous P2Y2 receptors, caused a similar 3-fold increase in calcium in both cell lines.

View larger version (26K): [in this window] [in a new window]   Fig. 3. Inositol phosphate production in response to NE in wild-type (1A-28) or mutant (208–210) 1A-ARs in stably transfected PC12 cells. Cells were labeled with [3H]myo-inositol (2 µCi/plate) for 1 to 2 days and production of total [3H]inositol phosphates stimulated by NE (100 µM) or UTP (100 µM) for 30 min was determined in the presence of 10 mM LiCl. **, p < 0.001; *, p < 0.05 compared with basal.

View larger version (20K): [in this window] [in a new window]   Fig. 4. Comparison of intracellular Ca2+ responses to NE with wild-type (1A-28) or mutant (208–210) 1A-ARs in stably transfected PC12 cells. Cells were loaded with fura-2 as described and stimulated with 100 µM NE at the arrow. Phentolamine (10 µM) and UTP (100 µM) were given at the indicated times. Shown is a single experiment, representative of three experiments with similar results. Over the three experiments, the effect of the mutation on the response to NE was highly significant (p < 0.001), whereas the effect of UTP was not statistically different.

MAPK Activation. We have previously shown that agonist stimulation of _1A-ARs in PC12 cells causes activation of several MAPK signaling pathways, including ERKs, JNKs, and p38 MAP kinase (Williams et al., 1998). The effect of NE, UTP, and NGF on MAPK activation in wild-type and ^208–210 _1A-ARs in PC12 cells is shown in Fig. 5. NE caused significant ERK activation in cells expressing the wild-type receptors, but no detectable activation in cells expressing the ^208–210 construct. UTP and NGF caused similar ERK activation in both cell lines (Fig. 5). More modest effects were observed on JNK activation, however, the effect of NE was still absent in the ^208–210 expressing cells.

View larger version (38K): [in this window] [in a new window]   Fig. 5. Activation of MAPKs by wild-type (1A-28) or mutant (208–210) 1A-ARs in stably transfected PC12 cells. Stably transfected cells were serum-starved for 2 h before adding vehicle (basal), 100 µM UTP, 100 µM NE, or 100 ng/ml NGF for 15 min and harvesting as described under Materials and Methods. Ten micrograms of protein was used for Western blotting with phospho-specific ERK1/2 antibody (top) or phospho-specific JNK antibody (bottom). The experiment shown is representative of three similar experiments. Using densitometry for semiquantitation of signals, the effect of UTP, NE, and NGF on ERK activation was highly significant (p < 0.01) in 1A-28 cells, and only the effect of NE was dramatically reduced in the mutant 208–210 cells. The effects on JNK activation were much weaker, but the effect of NE was reduced in the mutant 208–210 cells (p < 0.05). Total loading of ERKs and JNKs was determined in parallel and did not differ significantly between the samples (data not shown).

Activation of Pyk2. NE also increases tyrosine phosphorylation of Pyk2 in _1A-AR expressing PC12 cells (Zhong and Minneman, 1999a). Figure 6 shows that this effect is also blocked by the ^208–210 mutation.

View larger version (32K): [in this window] [in a new window]   Fig. 6. Activation of the tyrosine kinase Pyk2 by wild-type (1A-28) or mutant (208–210) 1A-ARs in stably transfected PC12 cells. Cells were treated with vehicle (basal) or 100 µM NE for 5 min, harvested, solubilized, and immunoprecipitated (IP) with an antibody to Pyk2. After electrophoresis and transfer, blots were Western blotted (IB) with an antibody specific for phosphotyrosine (P-Y). The experiment shown is representative of two similar experiments. In both experiments, NE caused at least a 3-fold increase in immunoreactivity for P-Y in 1A-28 cells, with no detectable increase in the mutant 208–210 cells.

Activation of Transcriptional Reporters. We previously used a variety of transcriptional reporters, consisting of repetitive DNA binding elements upstream of the luciferase gene, to characterize transcriptional effects of _1A-AR activation in PC12 cells (Minneman et al., 2000; Zhong et al., 2000). NE was found to activate reporters for AP1, SRE, CRE, NF-B, NFAT, GAS, and Stat in these cells (Minneman et al., 2000; Zhong et al., 2000). Figure 7 shows that none of these transcriptional responses were observed in PC12 cells expressing the ^208–210 uncoupled receptor, suggesting that they all require G protein activation.

View larger version (29K): [in this window] [in a new window]   Fig. 7. NE-stimulated luciferase activity in PC12 cells stably expressing wild type (1A-28) or mutant (208–210) 1A-ARs and transfected with different transcriptional reporters. Cells stably expressing wild-type (Cont) or mutant (Del) 1A-ARs were transfected with retroviral luciferase reporter constructs for AP1, SRE, CRE, NF-B, NFAT, GAS, or Stats as described previously (Minneman et al., 2000). NE (100 µM), vehicle, or positive controls [100 nM phorbol 12-myristate 13-acetate (PMA) for AP1, NF-B, and SRE reporters; 100 nM PMA + 100 nM ionomycin for NFAT; 10 µM forskolin for CRE; and 100 ng/ml interleukin 6 for GAS and Stat] were added to cells for 4 h before harvesting and performing luciferase measurements. The response to NE is expressed as a percentage of the response to the positive control and is the mean ± S.E.M. of four experiments performed in duplicate.

Effect of Overexpressing G16. Several reports have suggested that the Gq/11 family member G16 can promiscuously couple a variety of receptors to the phospholipase C/calcium signaling system. We determined whether overexpression of G16 could rescue the ^208–210 uncoupled receptor and reconstitute signaling. Figure 8 shows that we were able to successfully overexpress of G16 in PC12 cells expressing ^208–210 _1A-ARs; however, overexpression of this construct did not increase ERK or JNK activation by NE, or reconstitute the normal intracellular Ca²⁺ response to NE observed in cells expressing the wild-type receptor (Fig. 8).

View larger version (23K): [in this window] [in a new window]   Fig. 8. Effect of overexpression of G16 on responses to NE in 208–210 1A-AR expressing PC12 cells. PC12 cells stably expressing 208–210 1A-ARs were transfected with retrovirus coding for human G16 and zeocin resistance. Transfected cells were selected by exposure to 1 mg/ml zeocin for 2 weeks and G16 expression confirmed by Western blotting (top left). Stably selected cells were serum-starved, stimulated with vehicle (Basal), 100 µM NE, 100 ng/ml NGF, or 100 µM UTP for 15 min, and blotted with anti-phospho-ERK and anti-phospho-JNK antibodies as described in Fig. 5 (bottom left). Other cells were loaded with fura-2 and stimulated with NE (100 µM), phentolamine (10 µM), and UTP (100 µM) as indicated for measurement of intracellular calcium responses (right). Results are typical of two separate experiments.

Coimmunoprecipitation of G_q(EE) with FLAG-Tagged _1A- or ^208–210 _1A-AR. We determined whether ^208–210 _1A-ARs showed impaired G protein binding by comparing their ability to coimmunoprecipitate with G_q(EE) to that of the unmutated _1A-AR. G_q(EE) is a functionally normal mutant with an internal Glu-Glu epitope tag (Berlot, 1999). Receptor constructs containing N-terminal FLAG tags (Vicentic et al., 2002) were cotransfected with G_q(EE) into HEK293 cells. After membrane preparation and solubilization, FLAG-tagged receptors were immunoprecipitated with anti-FLAG affinity resin, eluted, run on SDS-PAGE, and blotted with anti-sera for G_q(EE). As shown in Fig. 9, similar coimmunoprecipitation of G_q(EE) was observed in cells cotransfected with either FLAG-_1A- or FLAG-^208–210_1A-ARs; however, no G_q(EE) was immunoprecipitated from cells transfected with this construct but no FLAG-tagged receptor. These data demonstrate that the ^208–210 _1A mutant retains its ability to interact with G_q, and implies that the deletion results in an inability to activate the G protein.

View larger version (37K): [in this window] [in a new window]   Fig. 9. Coimmunoprecipitation of Gq by FLAG-1A-AR and FLAG-208–2101A-AR in transfected HEK293 cells. HEK293 cells were cotransfected with Gq(EE) and FLAG-tagged receptor constructs or no receptor. After solubilization and anti-FLAG immunoprecipitation (IP), Gq(EE) immunoreactivity was detected in the solubilized fractions (top) and in the anti-FLAG immunoprecipitated complexes (bottom) by immunoblotting as described under Materials and Methods. The right lane shows that Gq(EE) cannot be immunoprecipitated by anti-FLAG affinity resin in cells transfected with Gq(EE) but no FLAG-tagged receptor.

	Discussion

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Agonist activation of GPCRs has traditionally been thought to cause a linear cascade of events caused by formation of intracellular second messengers. Recently, however, the interactions of GPCRs with accessory binding proteins, scaffolding proteins, and other signaling molecules have raised the possibility that GPCR activation may actually result in multiple signaling cascades, some of which may be independent of G protein activation and second messenger production. We have previously examined the effect of stably transfected _1A-AR activation in PC12 cells, and found that these receptors cause a variety of second messenger, mitogenic, and transcriptional responses (Zhong and Minneman, 1999a,c; Zhong et al., 2000). Some of these responses do not seem to be downstream of second messenger production (Berts et al., 1999; Zhong and Minneman, 1999a,c), suggesting that they may be G protein-independent. However, the results presented here now show that uncoupling of _1A-ARs from G protein activation by deletion of three amino acids in the third intracellular loop prevents all known responses to receptor activation. These include responses that are relatively proximal to receptor/G protein activation such as inositol phosphate formation and calcium release, more distal signals such as activation of the tyrosine kinase Pyk2 and activation of multiple MAPK pathways, and all nuclear transcriptional responses that are known to occur in response to receptor activation.

Because previous experiments showed that inhibitors of protein kinase C (GFX203290) and calcium mobilization (BAPTA) did not block _1A-AR-mediated MAP kinase activation in PC12 cells (Berts et al., 1999), we were somewhat surprised that these responses were blocked by the ^208–210 deletion. It is interesting that most other transcriptional responses to NE examined in these cells were either little changed (AP1 and SRE), or substantially increased (CRE and NFAT), by the presence of GFX203290 and BAPTA (Minneman et al., 2000). In fact, the only transcriptional response to NE that was largely inhibited by GFX203290 and BAPTA was the activation of the NF-B reporter construct (Minneman et al., 2000), suggesting that this is the only transcriptional response requiring mobilization of intracellular Ca²⁺ and activation of protein kinase C. The lack of effect of inhibitors of Ca²⁺ and protein kinase C on most of these responses suggests that MAPK and transcriptional responses to _1A-AR activation are often not linearly related to second messenger production in PC12 cells and raises the possibility that one or more of these responses might be independent of G protein activation. However, the fact that all of these transcriptional responses are completely eliminated by the ^208–210 mutation suggests that they may require similar structural determinants of agonist/receptor activation, whether or not they are downstream of traditional second messenger cascades.

The almost 10-fold increase in agonist affinity observed in the ^208–210 uncoupled _1A-AR is consistent with the uncoupled phenotype of this receptor. Although a similar increase was not observed with the analogous deletion in the hamster _1B-AR (Wu et al., 1995), there are many reports of increases (or decreases) in agonist binding affinity associated with uncoupling mutations in GPCRs. For example, deletion of a segment within the sixth hydrophilic segment of the hamster ₂-AR uncouples the receptor from Gs and increases agonist affinity (Strader et al., 1987). Similarly, an approximately 10-fold increase in affinity for epinephrine is observed with the Y348A mutation in hamster _1B-AR, which uncouples the receptor from downstream signaling pathways (Wang et al., 1997). Several other mutations in the intracellular loops of the hamster _1B-AR have also been shown to result in an increased agonist affinity (Greasley et al., 2001). However, these are not always correlated with an uncoupling phenotype, supporting previous observations that structural determinants of ligand binding and receptor activation are distinct.

These observations are consistent with the large body of evidence concerning the important role of the third intracellular loop of ₁-ARs in activation of Gq/11 signaling pathways (Greasley et al., 2001). Previous studies on other GPCRs have also stressed the importance of second intracellular loop, and the C or N terminus of the third intracellular loop in the efficiency of coupling and selectivity of G protein interactions (Wade et al., 1999; Greasley et al., 2001).

To demonstrate the selectivity of the ^208–210 deletion and determine the role of G protein activation in mitogenic and transcriptional responses, we attempted to rescue cells expressing the mutant receptors by overexpression of G16. The analogous deletion in the hamster _1B-AR had been shown to be uncoupled from Gq/11 and G14, but to show a weak coupling to heterologously expressed G16 (Wu et al., 1995). In fact, G16 has been shown to be relatively promiscuous in coupling GPCRs to signal activation and has been proposed to be a "universal" assay for GPCRs (Kostenis, 2001). Although we were able to obtain good heterologous expression of G16 in our PC12 cells, we did not observe any reconstitution of receptor-mediated second messenger or mitogenic responses with the ^208–210 deletion mutant. These data suggest either that G16 cannot effectively reconstitute these signaling pathways in PC12 cells because it does not couple effectively to downstream effectors or that the ^208–210 _1A-AR is not capable of activating this G protein. However, by using FLAG-tagged receptor constructs we demonstrated that the deletion mutant can directly interact with G_q in a manner similar to the wild-type construct. This suggests that the deletion results in an inability to activate the G protein, rather than blocking its ability to bind.

The observation that deletion of three amino acids in the third intracellular loop uncouples _1A-ARs from all second messenger, tyrosine kinase, MAPK, and transcriptional pathways in PC12 cells may suggest that all of these responses are downstream of G protein activation. It is possible, however, that this deletion might also influence other aspects of receptor function, and cause a similar uncoupling of receptors from non-G protein-mediated signaling pathways. However, if this were the case, then similar structural determinants in this small domain of the receptor must be required for any such G protein-independent signals. These observations demonstrate that all signals generated by agonist activation of _1A-ARs in PC12 cells are dependent on the sequence integrity of the N-terminal portion of the third intracellular loop, and probably require G protein activation.

	Footnotes

 
This study was supported by the National Institutes of Health.

DOI: 10.1124/jpet.103.050500.

ABBREVIATIONS: GPCR, G protein-coupled receptor; AR, adrenergic receptor; NE, norepinephrine; MAPK, mitogen-activated protein kinase; NGF, nerve growth factor; AP1, activator protein 1; SER, serum response element; cAMP response element; NF-B, nuclear factor-B; NFAT, nuclear factor of activated T cells; Stat, signal transducer and activator of transcription; HEK, human embryonic kidney; ¹²⁵I-BE, ¹²⁵I-BE 2254, 2-[-(4-hydroxyphenyl)-aminomethyl]tetralone; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH₂-terminal kinase; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; GAS, -interferon-activated sequence; GFX 203290, bisindolylmaleimide I.

Address correspondence to: Dr. Kenneth P. Minneman, Department of Pharmacology, Emory University Medical School, Atlanta, GA 30322. E-mail: kminneman{at}pharm.emory.edu

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