Receptor Selectivity of the Cloned Opossum G Protein-Coupled Receptor Kinase 2 (GRK2) in Intact Opossum Kidney Cells: Role in Desensitization of Endogenous {alpha}2C-Adrenergic but Not Serotonin 1B Receptors

Paola M. C. Lembo1, Mohammad H. Ghahremani and Paul R. Albert

Department of Pharmacology and Therapeutics (P.M.C.L., M.H.G.) McGill University Montreal, Quebec, Canada H3G 1Y6
Departments of Medicine and Cellular and Molecular Medicine (P.R.A.) Neuroscience Research Institute University of Ottawa Ottawa, Ontario, Canada K1H 8M5


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
To characterize the specificity of endogenously expressed G protein-coupled receptor kinases (GRKs) for endogenous Gi-coupled {alpha}2C-adrenergic and serotonin 1B (5-HT1B) receptors in the opossum kidney (OK) cell line, we have isolated a 3.073-kb OK-GRK2 clone encoding a 689-amino acid protein that shares 94.2% amino acid identity with rat GRK2. Northern blot analysis revealed the presence of GRK2 mRNA transcripts of 5.0 and 3.0 kb in OK cells. In intact OK cells, preincubation (45 min) with agonist (5-HT or UK 14304, 1 µM) reduced the maximal inhibition of forskolin-induced cAMP accumulation mediated by endogenous 5-HT1B and {alpha}2C-adrenergic receptors by 12 ± 2% or 17 ± 4%, respectively. In transfected OK cells overexpressing OK-GRK2, agonist-induced desensitization of the {alpha}2C-adrenergic receptor, but not the 5-HT1B receptor, was enhanced by 2- to 4-fold. Conversely, in cells overexpressing the kinase-inactive mutant OK-GRK2-K220R, {alpha}2C-adrenergic receptor desensitization was selectively abolished, whereas desensitization of the 5-HT1B receptor was slightly enhanced. Similarly, depletion of GRK-2 protein by stable transfection of full-length antisense OK-GRK2 cDNA blocked the desensitization of {alpha}2C-adrenergic receptors but not of 5-HT1B receptors. These results represent the first evidence of the coexistence of GRK2-dependent (for {alpha}2C receptors) and GRK2-independent (for 5-HT1B receptors) mechanisms of desensitization in intact cells and demonstrate the selectivity of GRK2 for distinct Gi-coupled receptors.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Many G protein-coupled receptor systems undergo a process of functional receptor desensitization in which receptor responsiveness is attenuated during prolonged exposure to stimuli such as hormones and neurotransmitters. Several mechanisms play pivotal roles in the functional desensitization of G protein-coupled receptors, particularly receptor phosphorylation that results in receptor-G protein uncoupling (1, 2). Other mechanisms that regulate receptor function include sequestration, which involves intracellular trafficking of receptors to endosomal compartments and ligand dissociation; and down-regulation, which entails a loss of receptor number due to enhanced receptor degradation or a decrease in receptor synthesis (3). Acute receptor desensitization appears to involve receptor-G protein uncoupling, which occurs rapidly and results in reduced efficacy of receptors to activate target G proteins (1, 2, 3). Uncoupling has been postulated to be mediated primarily by phosphorylation of the receptor, which can preferentially uncouple the receptor from some, but not necessarily all, signaling pathways (2, 4).

Acute receptor desensitization has historically been viewed as two separate mechanisms: heterologous and homologous desensitization. Heterologous desensitization is a process whereby activation of one type of receptor leads to the desensitization of nonstimulated receptors, whereas homologous desensitization refers to the loss of responsiveness of the stimulated receptor only. These rapid forms of desensitization appear to involve primarily the phosphorylation of the receptor on serine and threonine residues located in the intracellular cytoplasmic loops (1, 2, 3). There are at least two distinct classes of kinases involved in this phosphorylation process: the second-messenger kinases [e.g. protein kinase C (PKC), protein kinase A (PKA), and second-messenger independent receptor kinases termed G protein-coupled receptor kinases (GRKs) (2)].

GRKs are a family of serine/threonine kinases that phosphorylate G protein-coupled receptors in the ligand-activated state (5, 6). They belong to a family of kinases comprising six members (GRK1-GRK6) whose activities are differentially regulated by G protein ß{gamma}-subunits, phospholipids, or posttranslational modifications. Each kinase sequence has a centrally located catalytic domain flanked by an amino terminus that has conserved features and a carboxyl terminus that is highly variable. The C-terminal domain directs localization of the kinases to the membrane or receptor, by isoprenylation (via a CAAX box in GRK1), association with membrane-bound G-ß{gamma}-dimers (GRK2, GRK3), or by direct interactions with membrane phospholipids (GRK4, 6) (5, 6, 7, 8, 9). Mutations that inactivate either membrane translocation or kinase activity inhibit GRK-induced receptor phosphorylation and uncoupling (10, 11). The hallmark of these kinases is their specificity for activated (e.g. agonist-bound) receptors. Once phosphorylated, target receptors have an increased affinity for ß-arrestins, and the binding of ß-arrestin to the GRK-phosphorylated receptor is thought to prevent receptor coupling to G proteins (5, 6, 9). Thus, unlike receptor phosphorylation by PKC or PKA that directly uncouples the receptor, GRK-induced uncoupling requires both GRK and ß-arrestin molecules that recognize the appropriate receptor (9).

The regulation of G protein-coupled receptors by GRKs has been extensively documented in heterogeneous expression systems using overexpression of kinase and receptor cDNAs (5, 6, 7, 8, 9). Phosphorylation of receptors by GRKs has been shown to occur and regulate many G protein-coupled receptor systems such as ß2- and {alpha}2A-adrenergic receptors, m2 muscarinic acetylcholine receptors, rat olfactory receptor, and rhodopsin photoreceptor systems (9, 12, 13, 14, 15). While some substrate specificity is observed in these studies, the question remains whether the endogenous levels of GRK/ß-arrestin are sufficient to mediate agonist-induced homologous desensitization in nontransfected cells. Recently, GRK3 (ßARK2) was shown to selectively regulate and desensitize thrombin-mediated calcium mobilization in Xenopus oocytes (16) whereas GRK2 (ßARK1) and GRK3 were demonstrated to desensitize the inhibition of voltage-dependent calcium channels mediated by the {alpha}2-adrenergic receptor in isolated intact chick sensory neurons (17). In addition, Shih and Malbon (18) elegantly demonstrated that, depending on the heterologous expression system used, either the second messenger-dependent kinases (PKC or PKA) or the GRKs played a predominant role in promoting agonist-induced uncoupling of the ß2-adrenergic receptor. This raises the issue of physiological relevance in the modulation of receptors by these kinases. Hence, we decided to examine the influence of endogenous GRK on the homologous desensitization of serotonin 1B (5-HT1B) and {alpha}2C-adrenergic receptors endogenously expressed in the opossum kidney (OK) cell line (19, 20, 21, 22).

OK cells are an epithelial kidney cell line derived from the North American opossum, Didelphis virginiana (23). These cells endogenously express several G protein-coupled receptors including 5-HT1B, {alpha}2C-adrenergic, dopamine-D1, and PTH receptors (19, 21, 24, 25). These receptor subtypes each undergo agonist-promoted desensitization with respect to either inhibitory or stimulatory actions (20, 22, 26, 27). For example, both 5-HT1B and {alpha}2C-adrenergic receptors mediate inhibition of adenylyl cyclase in membrane preparations and undergo acute homologous desensitization of this response after sustained exposure to agonist (20, 22). We have addressed whether the desensitization observed can be detected in intact cell preparations by specifically measuring cAMP generation in whole cells.

The objectives of the present study were to determine whether 1) endogenous opossum GRKs were expressed in this cell line and 2) whether they play a role in the homologous desensitization process of the endogenously expressed G protein-coupled receptors. We have identified in OK cells a GRK-2 homolog, OK-GRK2,2 and shown that OK-GRK2 selectively mediated desensitization of the {alpha}2C-adrenergic receptor response, but did not interfere with desensitization of 5-HT1B receptor.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cloning of the Opossum GRK2 (OK-GRK2)
Degenerate oligonucleotide primers derived from the highly conserved catalytic region of GRKs were used to identify novel GRK cDNAs from the OK cell line (29). Two distinct 380-bp fragments (A1 and 10–1) of distinct nucleotide sequence were isolated that shared >92% predicted amino acid identity to the catalytic domains of rat GRK2 and GRK3. These cDNA fragments were used to clone full-length GRK cDNAs from a {lambda}-ZAP II cDNA library constructed using OK cell RNA. A 3.073-kb cDNA clone (named OK-GRK2) that shared 71.5% nucleotide identity with rat GRK2 cDNA sequence (Fig. 1Go) was identified in 10 of 10 positive clones. The OK-GRK2 cDNA contains a predicted open reading frame that encodes a 689-amino acid protein with 94.2% and 80.8% amino acid sequence identity with rat GRK2 and GRK3, respectively. The amino acid sequences of OK-GRK2 and rat GRK2 share 99% identity in the catalytic domain (Fig. 2Go, upper box), 90% in the amino terminus, 95% in the carboxyl terminus (Fig. 2Go, lower box); hence, the clone was designated OK-GRK2. In these domains amino acid substitutions were conservative (e.g. aliphatic-aliphatic); however, in less well conserved regions, nonconservative substitutions were observed.



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Figure 1. Nucleotide and Deduced Amino Acid Sequences of the OK-GRK2

The OK-GRK2 was isolated with a randomly labeled PCR fragment of OK-GRK2 cDNA (380 bp). The clone was 3.073 kb in length, numbered at the right. The amino acid sequence of OK-GRK2 begins with the first ATG in the nucleotide sequence and is numbered on the right-hand side of the sequence.

 


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Figure 2. Amino Acid Sequence Alignment of OK, Rat, and Bovine GRK2

The full amino acid sequence of OK-GRK2 (OK, top sequence) is compared with the rat (R, middle) and bovine (B, lower) GRK2 sequences, where conserved amino acids are represented as dashes and substitutions are as indicated. Overall amino acid identities of OK-GRK2 with the bovine GRK2 are 94.6% and 94.2% identity with the rat homolog. The sequences of the rat and bovine GRK2 were derived from GenBank. The amino acid sequences were aligned using the CLUSTAL algorithm program from DNASTAR Inc. (Madison, WI). The italicized and boxed regions of the alignment represent the catalytic (upper) and C-terminal (lower) domains.

 
Northern Blot Analysis of OK-GRK2
To verify the expression of OK-GRK2 RNA in this cell line, poly-A+ RNA isolated from OK cells was subjected to Northern blot analysis and probed with OK-GRK2 cDNA. As shown in Fig. 3Go, two distinct GRK-2-related mRNA species with molecular sizes of approximately 5.0 kb and 3.0 kb were detected. The intense band at 5.0 kb may correspond to the predominant OK-GRK2 cDNA clone in Fig. 1Go, whereas the smaller species may represent an alternatively spliced variant or use of an alternate polyadenylation site.



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Figure 3. Presence of GRK2 RNA Species in OK Cells

Poly A+ RNA (7 µg) from the OK cell line was isolated and separated on a 1.2% formaldehyde agarose gel, transferred to Hybond N membrane, and probed with the OK-GRK2 KpnI cDNA fragment (2.1 kb). Two bands of OK-GRK2 mRNA with estimated sizes of 5.0 and 3.0 kb were present after the autoradiogram was exposed for 4 days at -70 C. The hybridization and washing conditions were performed as described in Materials and Methods.

 
Receptor Selectivity of OK-GRK2
The role of endogenous OK-GRK2 in receptor desensitization of G protein-coupled receptors expressed endogenously in OK cells was addressed using three complementary approaches: 1) overexpressing wild-type OK-GRK2 to examine whether receptor attenuation is enhanced; 2) constructing and overexpressing a kinase-inactive mutant to compete with endogenous GRK2 (11); and 3) using a full-length antisense GRK2 cDNA construct to specifically deplete endogenous GRK2 protein expression (30). Site-directed mutagenesis was used to construct OK-GRK2-K220R, a mutant that lacks the catalytic lysine responsible for the phosphotransferase reaction (10). Stable transfectant colonies were selected by growth in G418-containing media, and clones with the highest level of RNA expression for each construct were isolated for Western blot analysis using antirecombinant bovine GRK2 polyclonal antibody (Fig. 4Go, A and B). As observed for in vitro-translated OK-GRK2 (data not shown), OK-GRK2 had the same mobility as purified bovine GRK2 (molecular mass, 80 kDa), but was weakly detected in nontransfected OK cells, suggesting that the endogenous level of OK-GRK2 is relatively low. Several clones for each construct were isolated for further characterization; however, only some of these maintained appropriate levels of expression during the course of experiments, including the overexpressed sense construct clones GRK-3 and GRK-18 and the kinase-inactive clone KI-5, which each displayed increased level of GRK expression compared with wild-type OK cells. The level of OK-GRK2 expression was greater in the GRK-18 clone than in GRK-3 cells (Fig. 4AGo). The antisense clone AS-4 was the only clone that maintained a depleted level of GRK2 expression as compared with the wild-type (W.T.) OK cells (Fig. 4BGo).



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Figure 4. Western Blot Analysis of OK-GRK2 in OK Cells

Wild-type, kinase-inactive mutant K220R and antisense GRK2 cDNA constructs were transfected stably in OK cells. Western blot analyses were performed using 50 mg/lane of cytosolic extracts from transfected clones and probed using antibovine GRK2 polyclonal antibody (1:1000). The arrows indicate the purified bovine GRK2 (2 ng), which migrated at Mr 80 kDa. A, Sense clones (GRK-3 and GRK-18) and kinase-inactive clone (KI-5) overexpress OK-GRK2 as compared with parental cell line (W.T.). B, Decreased amounts of OK-GRK2 were found in antisense clone (AS-4) as compared with the parental nontransfected cell line.

 
To establish a whole-cell assay for receptor desensitization, the ability of endogenous 5-HT1B receptors to undergo agonist-induced desensitization in wild-type OK cells was examined (Fig. 5Go). After examination of the time course of desensitization, cells were pretreated for 45 min using 1.0 to 1000 nM 5-HT, followed by a 12-min assay for receptor-mediated inhibition of forskolin-stimulated cAMP accumulation using a maximal concentration of 5-HT (1 µM). The agonist preincubation time of 45 min was chosen as a point of maximal acute desensitization, consistent with the half-time observed previously (20). The endogenous {alpha}2C receptors were also shown to undergo homologous desensitization in the parental OK cell line when pretreated with either 0.1 or 1 µM UK14304, a specific {alpha}2-adrenergic agonist (Table 1Go). In multiple assays, UK 14304 induced significant desensitization at both 0.1 and 1.0 µM concentrations, whereas 5-HT-induced desensitization was less potent (Table 1Go). The 5-HT1B and {alpha}2C receptors displayed a maximal level of desensitization of 12 ± 2 or 17 ± 4%, respectively, similar to the extent of desensitization obtained by others (20, 22). This assay was used to examine agonist-induced desensitization in the GRK clones.



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Figure 5. Agonist-Induced Desensitization of the 5-HT1B Receptor

Concentration dependence of 5-HT preincubation on inhibition of forskolin-stimulated cAMP accumulation. Cells were incubated with indicated concentrations of 5-HT for 45 min and assayed as previously described (31 ) for an additional 10-min incubation period using 1 µM 5-HT and 10 µM forskolin. The short (10 min) assay period allowed optimal detection of functional receptor desensitization. Values are means ± SEM.

 

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Table 1. Desensitization of 5-HT1B or {alpha}2C-Adrenergic Receptor-Mediated Inhibition of cAMP Accumulation in OK-GRK2 Cell Clones

 
The above experiments were repeated multiple times using several clones, and the data were averaged as relative desensitization as described in Materials and Methods (Table 1Go). In these experiments, no significant differences were observed among the clones compared with wild-type OK cells in forskolin-induced stimulation of cAMP level (-fold basal) or the extent of inhibition (% forskolin-stimulated cAMP level) by 5-HT or UK 14304 (1 µM) in vehicle-pretreated cells, except for the KI-5 clone (Table 1Go legend). This suggests that the clones retained receptor signaling properties equivalent to the wild-type OK cell line. The sense GRK-3 and GRK-18 clones that overexpress OK-GRK2 (Fig. 4AGo) displayed enhanced desensitization of the {alpha}2C-adrenergic response by 2- to 4-fold compared with wild-type OK cells (P < 0. 02 for GRK-3 and P < 0. 002 for GRK-18 at both concentrations of UK 14304). By contrast, desensitization of the 5-HT1B-induced response in the GRK clones was similar to OK cells except for the GRK-18 clone that more strongly expresses GRK2, in which a slight enhancement of desensitization compared with OK cells was observed at 1.0 µM 5-HT (P < 0. 02). Although the extent of desensitization at 0.1 µM 5-HT achieved significance (P < 0.05) in the GRK-18 clone, but was not significantly different from that in wild-type OK cells. Thus, the 5-HT1B response was much less sensitive to overexpression of OK-GRK2 than the {alpha}2C-induced response. In the kinase-inactive mutant clone KI-5, desensitization of {alpha}2C receptors by 0.1 and 1.0 µM UK 14304 was completely abrogated and was significantly reduced compared with OK cells (P < 0. 05). By contrast, the desensitization of the 5-HT1B response appeared unchanged or slightly enhanced. The KI-5 clone displayed a significant desensitization of the 5-HT1B receptor at 0.1 µM 5-HT (P < 0. 05) that was not present in the wild-type OK cells (Table 1Go), suggesting a slight enhancement in the potency of 5-HT for desensitization. Another GRK2-K220R-positive clone, KI-19 (n = 3) had a similar profile as KI-5 at both concentrations of agonist. The desensitization of {alpha}2C receptors was reduced by >50% as compared with the parental cell line, but 5-HT1B receptor desensitization was unaffected (data not shown); however, with increased passage in culture this response and mutant GRK2 expression in this clone were lost. In the AS-4 clone in which endogenous OK-GRK2 protein level was reduced (Fig. 4BGo), agonist-mediated desensitization of the {alpha}2C-adrenergic receptor was abolished at 0.1 µM UK14304, although a small but significant desensitization occurred at 1.0 µM (P < 0. 05); at this concentration, desensitization was clearly inhibited compared with OK cells (P < 0. 01; Table 1Go). These observations indicate that the {alpha}2C receptors are regulated by OK-GRK2, whereas regulation of 5-HT1B receptor desensitization involves primarily GRK-independent processes.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A Novel Opossum GRK
The opossum GRK clone we describe is most similar to the previously identified bovine and rat GRK2 (ßARK1) (32, 33); hence we have designated it opossum GRK2. Computer alignment revealed considerable nucleotide identity of the coding region of OK-GRK2 to the rat and bovine GRK2 cDNAs (83.8% and 86.3%, respectively) that was lower than the homology of rat and bovine nucleotide sequences (89.6% identical). Similarly, the deduced amino acid sequence of OK-GRK2 shares 94.2% and 94.6% identity with rat and bovine sequences, respectively, reflecting the evolutionary distance between marsupial and mammalian species. By contrast, the rat and bovine species share 98.3% amino acid identity. Important structural domains, such as the catalytic domain, were highly conserved in GRK2 homologs. For example, there is more than 99% amino acid identity in the catalytic domain among these three species of GRK2. In addition, the C-terminal region containing the pleckstrin homology domain that is responsible for the docking of ß{gamma}-subunits is highly conserved (34, 35). Thus, the clone has the structural characteristics of a functional GRK.

Northern blot analysis revealed two mRNA transcripts of approximately 5.0 and 3.0 kb. Similarly, the message size we observe was approximately the same as found in the bovine brain (32). The presence of the second smaller band at 3.0 kb might represent alternative splicing of a large intron in the untranslated regions of the mRNA or alternate usage of 3'-polyadenylation sites. The gene for the human GRK2 was recently cloned and was found to be composed of 21 exons interrupted by 20 introns (36). If this structural complexity is conserved in the opossum GRK2 gene, it may explain the smaller mRNA seen at 3.0 kb.

Receptor Specificity of OK-GRK2
Bylund and co-workers have shown that homologous desensitization of endogenously expressed 5-HT1B and {alpha}2C-adrenergic receptors occurs in intact OK cells with approximate half-times of 60 or 30 min, respectively (20, 22). Using a 45-min agonist preincubation period, we detected a similar extent of desensitization of these receptors in intact OK cells. Although rapid desensitization via receptor uncoupling occurs within minutes and is rapidly reversible, GRK-mediated desensitization has a slower time course and may involve both uncoupling and receptor internalization, both processes that are potentially regulated by GRK (37). It is unlikely that receptor down-regulation is involved, since this requires a longer time course and higher agonist concentration (3, 20).

Using this desensitization paradigm our observations demonstrate a Gi-coupled receptor specificity of OK-GRK2 in the OK cell line. Overexpression of OK-GRK2 enhanced agonist-induced desensitization of the {alpha}2C-adrenergic receptors at 1 µM UK14304, whereas minimal enhancement of 5-HT1B receptor desensitization was observed only in the GRK-18 clone that more strongly overexpressed OK-GRK2. Thus, the cloned OK-GRK2 is functionally active and potent in mediating {alpha}2C receptor desensitization, but only weakly influences desensitization of the 5-HT1B receptor at the highest level of expression. The inhibitory action of mutant or antisense GRK2 expression suggests the importance of the apparently low level of endogenous OK-GRK2 in {alpha}2C-adrenergic receptor desensitization. Kinase-deficient GRK2 mutants have been shown to preclude agonist-induced desensitization of several receptor subtypes, including endogenously expressed ß2-adrenergic receptors (but not for PGE2 receptors) in bronchial cells (10), and the acute desensitization of m2-muscarinic or angiotensin II receptors in transfected HEK-293 cells (13, 38). Our results indicate that the kinase-inactive OK-GRK2-K220R mutant prevents the desensitization of the endogenously expressed {alpha}2C-adrenergic receptor, but not the 5-HT1B receptor. GRK2 has been shown to bind to ß{gamma}-subunits of G proteins and to phosphatidyl inositol bisphosphate via its pleckstrin homology domain (34, 35, 39). OK-GRK2-K220R might sequester B{gamma}-subunits to prevent GRK2 from translocation to the receptor, thus blocking its function. Alternately, kinase-inactive GRK2 may interact directly with phosphorylation sites on the receptor to sterically hinder access of endogenous GRKs, including OK-GRK2. Thus, overexpression of kinase-inactive GRK2 could block the action of multiple GRKs. Depletion of OK-GRK2 using antisense RNA expression resulted in a loss of detectable OK-GRK2 protein and a complete attenuation of agonist-mediated desensitization of {alpha}2C-adrenergic receptors. These results imply that, under physiological levels of expression, {alpha}2C-adrenergic receptor is selectively regulated by OK-GRK2, consistent with the presence of several conserved phosphorylation sites for GRK in the third cytoplasmic loop (21). The opossum {alpha}2C-adrenergic receptor is a homolog of the human {alpha}2C4 subtype (21), which has never been shown to undergo homologous desensitization (12). Taken together, these data provide compelling evidence for functional activity of OK-GRK2 in the agonist-induced desensitization of the endogenous {alpha}2C-adrenergic receptor in OK cells. In the same cells, OK-GRK2 did not substantially modulate desensitization of the 5-HT1B receptor.

The relative insensitivity of 5-HT1B receptor desensitization to modulation of GRK2 activity indicates that at physiological and moderately supraphysiological levels of GRK2, desensitization of the 5-HT1B receptor proceeds by a GRK2-independent mechanism. Concurrent observation of GRK2-mediated desensitization of the {alpha}2C receptor in the same OK clones indicates that GRK2 is active and selective for the {alpha}2C receptor at the GRK2 protein level found in intact cells. These results suggest that factors (e.g. localization or accessibility, other regulatory proteins) additional to suitability as a phosphorylation substrate may determine sensitivity to GRK2. Alternately, it may be that the 5-HT1B receptor is a weaker substrate in vitro for GRK2 than the {alpha}2C-receptor.

Protein kinases other than OK-GRK2 may mediate agonist-induced 5-HT1B receptor desensitization. Recently, it was shown that the dopamine D1A receptor was differentially regulated in terms of agonist-mediated desensitization by various GRKs when transfected in HEK-293 cells (40). The notion that another member of the GRK family participates in agonist-promoted attenuation of the 5-HT1B receptor is conceivable. A 380-bp GRK3-related cDNA fragment was identified in the OK cell line using PCR with degenerate oligonucleotides and could correspond to a second GRK subtype. This suggests that other GRKs might be expressed in the cell line that may be responsible for promoting homologous desensitization of the 5-HT1B receptor. This could be investigated using specific GRK monoclonal antibodies to determine GRK specificity as elegantly demonstrated by Oppermann and colleagues (41). However, the desensitization of the 5-HT1B receptor in the kinase-inactive clones suggests that other GRK subtypes that are susceptible to competition with the kinase-inactive mutant do not mediate this response. Another possibility is that the attenuation response for the 5-HT1B receptors may not necessarily be mediated by GRK-induced phosphorylation, but by a second messenger kinase such as PKC. Interestingly, the 5-HT1B receptor-mediated calcium response was shown to be uncoupled by acute preactivation of PKC in OK cells (42).

The role of GRK in vivo may supercede its role in receptor desensitization. Recently, transgenic mice overexpressing GRK2 specifically in cardiac tissue were generated. These mice not only exhibited reduced cardiac contractility in response to isoproterenol but the myocardial adenylyl cyclase activity was attenuated due to the reduced functional coupling of the ß2-adrenergic receptors (43). In contrast, mice overexpressing a GRK inhibitor displayed increased cardiac contractility. In addition, mice with a disruption of the GRK2 gene were shown to display severe cardiac malformations, suggesting a role in fetal development (44). These results suggest a pivotal role played by GRK2 physiologically.

To conclude, we have cloned a functional GRK2 from the opossum kidney cell line and have shown that in intact cells OK-GRK2 displays Gi-coupled receptor specificity in mediating the homologous desensitization of endogenous {alpha}2C-adrenergic receptors, but not of 5-HT1B receptors. Nevertheless, the 5-HT1B receptor is phosphorylated in vitro by OK-GRK, suggesting the other factors determine receptor susceptibility to GRK-mediated desensitization.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Restriction endonucleases and other molecular biology reagents were purchased from Boehringer Mannheim (Indianapolis, IN) except as indicated. Sequenase was from Pharmacia (Piscataway, NJ); [{alpha}-32P]ATP and [{tau}-32P]ATP were purchased from Amersham Corp. (Arlington Heights, IL). Serotonin, 3-isobutyl-1-methylxanthine, and forskolin were from Sigma Chemical Co. (St. Louis, MO) and UK 14304 was purchased from RBI Chemicals (Natick, MA). Tissue culture media and sera were from GIBCO-BRL (Gaithersburg, MD), and OK cells were purchased from the ATCC (Manassas, VA).

Cell Culture
OK cells were grown to 80–90% confluence with DMEM (Hi-glucose) and supplemented with 8% FBS at 37 C in a humidified atmosphere with 5% carbon dioxide. Media were changed 12–24 h before experimentation.

PCR
Random hexamers were used as primers to reverse transcribe total RNA (1 µg) isolated from the OK cell line using Superscript reverse-transcriptase (GIBCO-BRL). The cDNAs were amplified by PCR [1 min at 95 C, 2 min at 54 C, 2 min at 72 C for 40 cycles] using degenerate oligonucleotides, sense (5'-GGCAAGATGTA(T/C)GC-(A/T/C/G)ATGAA-3') and antisense (3'-AC(C/T)TCGGG(A/C/G/T)GCCATGTACCC-5') designed to highly conserved regions of the catalytic domain of GRK2 [Drosophila, rat, and bovine (29)]. The PCR reactions contained 1 µg of reverse-transcribed OK cell mRNA, 1.5 mM MgCl2, and 5x Hot Tub buffer (Amersham). Two distinct 380-bp cDNAs were isolated (10–1 and A-1) and subcloned into the EcoRV site of pBluescript (KS+, Stratagene, La Jolla, CA). DNA sequence analysis using the Sanger method with a T7 polymerase-based DNA sequencing kit (Pharmacia) revealed that they shared 81% and 66% identity, respectively, at the nucleotide level to the rat GRK2.

Construction of OK cDNA Library
Poly-A+ RNA was isolated from the OK cell line using the QuickPrep Micro mRNA Purification kit (Pharmacia). For cDNA synthesis, 5 µg of Poly-A+ RNA was reverse-transcribed using Superscript reverse-transcriptase, and the complete synthesis of double-stranded DNA was performed using the oligo-dT Riboclone cDNA Synthesis kit (Promega, Madison, WI). The blunt-ended cDNAs were fractionated and EcoRI adapters were ligated followed by ligation into EcoRI-predigested Lambda ZAP II arms (Stratagene). Gigapack II Gold (Stratagene) extracts were used to package recombinant {lambda}-phage extracts.

cDNA Cloning
The oligo-dT-primed OK cell cDNA library was amplified and 106 plaques were plated, transferred to Hybond N+ membranes, and screened with the 350-bp A-1 and 10–1 PCR fragments labeled using a randomly primed labeling kit (Boehringer-Mannheim) with the following hybridization conditions: 50% formamide, 50% dextran-sulfate, 1% SDS, 5x NaCl-sodium citrate (SSC), 5x Denhardt’s, 10 mM Tris (solution A), and 100 µg/ml sonicated salmon sperm DNA at 42 C overnight. The filters were washed with 2x SSC/1% SDS at room temperature followed by a high-stringency wash with 0.1% SDS/0. 2x SSC at 65 C. The 10 clones identified by this method were isolated by repeated plating and screening with the labeled PCR fragments. The isolated clones were rescued with helper phage to yield the cDNAs as inserts in pBluescript SK vector. All 10 clones were restriction mapped and were found to be identical. One clone was subjected to manual and automated sequencing in sense and antisense orientations using T7 polymerase and oligonucleotides directed to regions of the sequence, or reverse and universal primers. This 3.073-kb clone was highly homologous to rat GRK2 and was therefore named OK-GRK2.

Northern Blot Analysis
Poly-A+ RNA was isolated from the OK cell line using the QuickPrep Micro mRNA Purification kit (Pharmacia). Seven micrograms of poly-A+ OK RNA and 3 µg of 0.24–9.5 kb RNA ladder (GIBCO-BRL) were fractionated on a 1. 3% agarose-formaldehyde gel and transferred to Hybond-N membrane (Amersham). The 2.1 kb KpnI fragment of OK-GRK2 was labeled and hybridized to the membrane in solution A overnight with 100 µg/ml sheared, sonicated salmon sperm DNA at 42 C. The filter was washed with 0.1% SDS/0.2x SSC at 65 C and exposed for 4 days at -70 C to Kodak XAR film (Eastman Kodak, Rochester, NY) with an intensifier.

Construction of K220R Mutant and Stable Transfections of Sense, Antisense, and K220R Mutant in OK Cells
The EcoRI fragment of the OK-GRK2 gene was subcloned into pSelect to use as a template for site-directed mutagenesis (Altered-sites mutagenesis, Promega). The oligonucleotide 5'-ATCAAGACACCTCATGGCATA-3' was used to incorporate the point mutation. The mutation was confirmed by DNA sequencing using T7 polymerase (Pharmacia). The K220R mutant, antisense, and wild-type OK-GRK2 cDNAs were subcloned into the eukaryotic expression vector pcDNA-3 (Invitrogen, San Diego, CA) and stably transfected into OK cells using the calcium phosphate coprecipitation method. Geneticin-resistant clones were selected and grown in DMEM supplemented with 10% FCS and 1.5 mg/ml Geneticin. Positive clones were identified by Northern and Southern blot analyses.

Western Blot Analysis
Western blot analyses were performed using cytosolic fractions from the RNA-positive transfected clones. To prepare cytosolic extracts, OK cells and clones were trypsinized and resuspended in 150 µl of lysis extraction buffer [150 mM NaCl, 50 mM Tris-HCl (pH 7. 8), 0. 01% sodium azide, 10 mM NaF, 30 mM sodium pyrophosphate] followed by addition of 150 µl of lysis extraction buffer containing 4% NP-40, 5 mM Na3VO4, 10 µg/ml leupeptin, 10 mM benzamidine, 2 µg/ml soybean trypsin inhibitor, 1 mg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 10 mM N-tosyl-1-phenylalanine chloromethyl ketone, and 20 mM iodoacetamide. This was incubated on ice for 30 min, and the supernatant was recovered after centrifugation at 4 C, 15 min, at 12,000 x g. Using BSA as a standard (Bio-Rad, Richmond, CA) for protein concentration, 50 µg of total protein were loaded in SDS-PAGE sample buffer, resolved by a 10% SDS-PAGE, and electroblotted onto ECL nitrocellulose membranes (Western blotting ECL kit, Amersham) for 2 h at 250 mA constant current. Blots were preblocked in TBS-Blotto (150 mM NaCl, 20 mM Tris-HCl, pH 8.0, 5% nonfat dried milk) at room temperature. The recombinant bovine GRK2 polyclonal antibody (1:1000) was hybridized to the membranes in TBS-Blotto for 1 h at room temperature. Blots were washed in TBS-Blotto-0.5% Tween 20 (4 x 15 min, 3 x 5 min for high stringency) or TBS-Blotto-0.05% Tween 20 (4 x 15 min, or 3 x 5 min for low stringency) and incubated with secondary antibody (horseradish peroxidase-conjugated antirabbit IgG) 1:1000 for 1 h at room temperature. Blots were washed in TBS-Blotto-Tween 20 as described above and incubated with the Amersham ECL detection reagents and developed for up to 10 min.

cAMP Assay
Measurement of cAMP was performed as described previously with some modifications (31). Briefly, OK cells were plated in 24-well dishes and propagated to near confluence for assay. The cells were washed twice (5 min each) with 1 ml of DMEM/HEPES (serum-free DMEM + 20 mM HEPES, pH 7.2) and incubated with 0. 5 ml/well of DMEM/HEPES for 45 min containing either no addition, 100 nM/1 µM 5-HT, or 100 nM/1 µM UK 14304. Typically, half of each plate was treated with 5-HT and half with UK 14304 to measure actions of both compounds under identical conditions. After incubation, medium was quickly decanted and replaced with warm assay medium (DMEM/HEPES + 100 µM 3-isobutyl-1-methylxanthine) with agents (10 µM forskolin, 1 µM 5-HT, or 1 µM UK14304) and incubated for 15 min at 37 C. The 15-min assay period was the minimal time to process in parallel multiple plates of OK cells and clones. In control experiments, the cells were washed twice with DMEM/HEPES before addition of assay medium: it was determined that the 5-HT pretreatment did not alter subsequent basal or forskolin-stimulated cAMP levels. The reactions were stopped by removing the medium. The supernatants were collected and assayed for cAMP by a specific RIA (ICN) as described (31). Standard curves displayed an average IC50 value of 0.5 ± 0.2 pmol using cAMP as standard. Data for cAMP assays are presented as mean ± SEM for triplicate wells. Percent desensitization was calculated as follows {1 - [(F - XFA)/(F - FA)]} x 100, where cAMP level was determined under the following conditions: F = forskolin, FA = forskolin + agonist; XFA = forskolin + agonist after the indicated agonist pretreatment.


    ACKNOWLEDGMENTS
 
We are grateful to Dr. J. L. Benovic for providing the purified bovine GRK2 and the recombinant polyclonal antibody. We thank Marc Pinard for critical analysis of the manuscript, Dr. M. Szyf for helpful discussions, and Christine Forget for expert technical assistance.


    FOOTNOTES
 
Address requests for reprints to: Dr. Paul R. Albert, Departments of Medicine and Cellular and Molecular Medicine, Neuroscience Research Institute, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5. E-mail: palbert{at}uottawa.ca

This work was supported by the Medical Research Council (MRC), Canada. P.R.A. is recipient of the Novartis/MRC Michael Smith Chair in Neuroscience.

1 Present address: Astra Research Centre, 7171 Frederick Banting, Ville St. Laurent, Canada H4S-1Z9. Back

2 The GenBank accession number for OK-GRK2 cDNA is AF087455. Back

Received for publication July 31, 1998. Revision received August 28, 1998. Accepted for publication September 28, 1998.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Collins S, Caron MG, Lefkowitz RJ 1991 Regulation of adrenergic receptor responsiveness through modulation of receptor gene expression. Annu Rev Physiol 53:497–508[CrossRef][Medline]
  2. Lohse MJ 1993 Molecular mechanisms of membrane receptor desensitization. Biochim Biophys Acta 1179:171–188[Medline]
  3. Hein L, Kobilka BK 1995 Adrenergic receptor signal transduction and regulation. Neuropharmacology 34:357–366[CrossRef][Medline]
  4. Lembo PMC, Albert PR 1995 Multiple phosphorylation sites are required for pathway-selective uncoupling of the 5-HT1A receptor by protein kinase C. Mol Pharmacol 48:1024–1029[Abstract]
  5. Premont R, Inglese J, Lefkowitz RJ 1995 Protein kinases that phosphorylate activated G protein-coupled receptors. FASEB J 9:175–182[Abstract/Free Full Text]
  6. Inglese J, Freedman NJ, Koch WJ, Lefkowitz RJ 1993 Structure and mechanisms of the G protein-coupled receptor kinases. J Biol Chem 268:23735–23738[Free Full Text]
  7. Stoffel RH, Randall RR, Premont RT, Lefkowitz RJ, Inglese J 1994 Palmitolylation of G protein-coupled receptor kinase, GRK6. J Biol Chem 269:27791–27794[Abstract/Free Full Text]
  8. Premont RT, Macrae AD, Stoffel RH, Chung N, Pitcher JA, Ambrose C, Inglese J, MacDonald ME, Lefkowitz RJ 1996 Characterization of the G protein-coupled receptor kinase, GRK4. J Biol Chem 271:6403–6410[Abstract/Free Full Text]
  9. Sterne-Marr R, Benovic JL 1995 Regulation of G protein-coupled receptors by receptor kinases and arrestins. Vitam Horm 51:193–234[Medline]
  10. Kong G, Penn R, Benovic JL 1996 A ßadrenergic receptor kinase dominant negative mutant attenuates desensitization of the ß2-adrenergic receptor. J Biol Chem 269:13084–13087[Abstract/Free Full Text]
  11. Pitcher JA, Inglese J, Higgins JB, Arriza JL, Casey PJ, Kim C, Benovic JL, Kwatra MM, Caron MG, Lefkowitz RJ 1992 Role of beta gamma subunits of G proteins in targeting the beta-adrenergic receptor kinase to membrane-bound receptors. Science 257:1264–1267[Medline]
  12. Kurose H, Lefkowitz RJ 1994 Differential desensitization and phosphorylation of three cloned and transfected {alpha}2-adrenergic receptor subtypes. J Biol Chem 269:10093–10099[Abstract/Free Full Text]
  13. Pals-Rylaarsdam R, Yirong X, Witt-Enderby P, Benovic JL, Hosey MM 1995 Desensitization and internalization of the m2 muscarinic acetylcholine receptor are directed by independent mechanisms. J Biol Chem 270:29004–29011[Abstract/Free Full Text]
  14. Dawson TM, Arriza J, Jaworsky DE, Borisy FF, Attramadal H, Lefkowitz RJ, Ronnett GV 1993 ßadrenergic receptor kinase-2 and ß-arrestin-2 as mediators of odorant-induced desensitization. Science 259:825–829[Medline]
  15. Kawamura S 1993 Rhodopsin phosphorylation as a mechanism of cyclic GMP phosphodiesterase regulation by S-modulin. Nature 362:855–857[CrossRef][Medline]
  16. Ishii K, Chen J, Ishii M, Koch WJ, Freedman NJ, Lefkowitz RJ, Coughlin SR 1994 Inhibition of thrombin signaling by a G protein-coupled receptor kinase: functional specificity among G protein-coupled receptor kinases. J Biol Chem 269:1125–1130[Abstract/Free Full Text]
  17. Diverse-Pierluissi M, Inglese J, Stoffel RH, Lefkowitz RJ, Dunlap K 1996 G protein-coupled receptor kinase mediates desensitization of norepinephrine-induced calcium channel inhibition. Neuron 16:579–585[Medline]
  18. Shih M, Malbon CC 1994 Oligodeoxynucleotides antisense to mRNA encoding protein kinase reveal distinctive cell-type-specific roles in agonist-induced desensitization. Proc Natl Acad Sci USA 91:12193–12197[Abstract/Free Full Text]
  19. Cerutis DR, Hass NA, Iversen LJ, Bylund DB 1994 The cloning of an OK cell cDNA encoding a 5-hydroxytryptamine 1B receptor. Mol Pharmacol 45:20–28[Abstract]
  20. Pleus RC, Bylund DB 1992 Desensitization and down-regulation of the 5-hydroxytryptamine1B receptor in the opossum kidney cell line. J Pharmacol Exp Ther 261:271–277[Abstract]
  21. Blaxall HS, Cerutis DR, Hass NA, Iversen LJ, Bylund DB 1994 Cloning and expression of the {alpha}2C-adrenergic receptor from the OK cell line. Mol Pharmacol 45:176–181[Abstract]
  22. Jones SB, Leone SL, Bylund DB 1990 Desensitization of the a-adrenergic receptor in HT29 and opossum kidney cell lines. J Pharmacol Exp Ther 254:294–300[Abstract]
  23. Koyama H, Goodpasture C, Miller MM, Teplitz RL, Riggs AD 1978 Establishment and characterization of a cell line from the American opossum Didelphys Virginiana. In Vitro 14:239–246[Medline]
  24. Nash SR, Godinot N, Caron MG 1994 Cloning and characterization of the opossum kidney cell D1 dopamine receptor: expression of identical D1A and D1B dopamine receptor mRNAs in opossum and brain. Mol Pharmacol 44:918–925[Abstract]
  25. Jüppner H, Abou-Samra AB, Freeman MW, Kong XF, Schipani S, Richards J, Kolakowski LF, Hock JT, Kronenberg HM, Segre GV 1991 A G protein-linked receptor for parathyroid hormone and parathyroid hormone-related peptide. Science 254:1024–1026[Medline]
  26. Bates MD, Caron MG, Raymond JR 1991 Desensitization of DA1 dopamine receptors coupled to adenylyl cyclase in opossum kidney cells. Am J Physiol 260:F937–F945
  27. Fujimori A, Miyauchi A, Hruska KA, Martin KJ, Avioli LV, Civitelli R 1993 Desensitization of calcium messenger system in parathyroid hormone-stimulated opossum kidney cells. Am J Physiol 262:E918–E924
  28. Ng GY, George SR, Zastawny RL, Caron M, Bouvier M, Dennis M, O’Dowd BF 1993 Human serotonin1B receptor expression in Sf9 cells: phosphorylation, palmitoylation, and adenylyl cyclase inhibition. Biochemistry 32:11727–11733[Medline]
  29. Cassill JA, Whitney M, Joazeiro CAP, Becker A, Zuker CS 1991 Isolation of Drosophila genes encoding G protein-coupled receptor kinases. Proc Natl Acad Sci USA 88:11067–11070[Abstract]
  30. Albert PR, Morris SJ 1994 Antisense knockouts: molecular scalpels for the dissection of signal transduction. Trends Pharmacol Sci 15:250–254[CrossRef][Medline]
  31. Liu YF, Albert PR 1991 Cell-specific signalling of the 5-HT1A receptor. Modulation by PKC and PKA. J Biol Chem 266:23689–23697[Abstract/Free Full Text]
  32. Benovic JL, DeBlasi A, Stone WC, Caron MG, Lefkowitz JR 1989 ßadrenergic receptor kinase: primary structure delineates a mutigene family. Science 246:235–240[Medline]
  33. Arriza JL, Dawson TM, Simerly RB, Martin LJ, Caron MG, Snyder SH, Lefkowitz RJ 1992 The G protein-coupled receptor kinases bARK1 and bARK2 are widely distributed at synapses in rat brain. J Neurosci 12:4045–4055[Abstract]
  34. Neer EJ, Schmidt CJ, Nambudripad R, Smith TF 1994 The ancient regulatory-protein family of WD-repeat proteins. Nature 371:297–300[CrossRef][Medline]
  35. Inglese J, Koch WJ, Touhara K, Lefkowitz RJ 1995 G beta gamma interactions with PH domains and Ras-MAPK signaling pathways. Trends Biochem Sci 20:151–156[CrossRef][Medline]
  36. Penn RB, Benovic JL 1994 Structure of the huan gene encoding the beta-adrenergic receptor kinase. J Biol Chem 269:14924–14930[Abstract/Free Full Text]
  37. Ferguson SS, Barak LS, Zhang J, Caron MG 1996 G protein-coupled receptor regulation: role of G protein-coupled receptor kinases and arrestins. Can J Physiol Pharmacol 74:1095–1110[CrossRef][Medline]
  38. Oppermann M, Freedman NJ, Alexander RW, Lefkowitz RJ 1996 Phosphorylation of the type 1A Angiotensin receptor by G protein-coupled receptor kinases and protein kinase C. J Biol Chem 271:13266–13272[Abstract/Free Full Text]
  39. Pitcher JA, Touhara K, Sturgis-Payne E, Lefkowitz RJ 1995 Pleckstrin homology domain-mediated membrane association and activation of the ßadrenergic receptor kinase requires coordinate interaction with Gbg subunits and lipid. J Biol Chem 270:11707–11710[Abstract/Free Full Text]
  40. Tiberi M, Nash SR, Bertrand L, Lefkowitz RJ, Caron MG 1996 Differential regulation of dopamine D1A receptor responsiveness by various G protein-coupled receptor kinases. J Biol Chem 271:3771–3778[Abstract/Free Full Text]
  41. Oppermann M, Diversepierluissi H, Drazner MH, Dyer SL, Freedman NJ, Peppel KC, Lefkowitz RJ 1996 Monoclonal antibodies reveal substrate specificity among G protein-coupled receptor kinases. Proc Natl Acad Sci USA 93:7649–7654[Abstract/Free Full Text]
  42. Lembo P, Albert PR 1994 5-HT1B receptors mediate stimulatory calcium signaling opossum kidney OK cells: negative regulation by protein kinase C. Can J Physiol Pharmacol 72[Suppl 1]:536
  43. Koch WJ, Rockman, HA, Samama, P, Hamilton, R, Milano, CA, Lefkowitz, RJ 1995 Cardiac function in mice over-expressing the ß-adrenergic receptor kinase or a GRK inhibitor. Science 268:1350–1353[Medline]
  44. Jaber M, Koch WJ, Rockman H, Smith B, Bond RA, Sulik KK, Ross Jr J, Lefkowitz RJ, Caron MG, Giros B 1996 Essential role of beta-adrenergic receptor kinase 1 in cardiac development and function. Proc Natl Acad Sci USA 93:12974–12979[Abstract/Free Full Text]