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【关键词】 Characterization
To analyze the interaction between the neurokinin-1 (NK-1) receptor and G-protein coupled receptor kinases (GRKs), we performed bioluminescence resonance energy transfer2 (BRET2) measurements between the family A NK-1 receptor and GRK2 and GRK5 as well as their respective kinase-inactive mutants. We observed agonist induced interaction of both GRK5 and GRK2 with the activated NK-1 receptor. In saturation experiments, we observed GRK5 to interact with the activated receptor in a monophasic manner while GRK2 interacted in a biphasic manner with the low affinity phase corresponding to receptor affinity for GRK5. Agonist induced GRK5 interaction with the receptor was dependent on intact kinase-activity, whereas the high affinity phase of GRK2 interaction was independent of kinase activity. We were surprised to find that the BRET2 saturation experiments indicated that before receptor activation, the full-length NK-1 receptor, but not a functional C-terminal tail-truncated receptor, is preassociated with GRK5 in a relatively low-affinity state. We demonstrate that GRK5 can compete for agonist induced GRK2 interaction with the NK-1 receptor, whereas GRK2 does not compete for receptor interaction with GRK5. We suggest that GRK5 is preassociated with the NK-1 receptor and that GRK5, rather than GRK2, is a key player in competitive regulation of GRK subtype specific interaction with the NK-1 receptor.
Seven transmembrane (7TM) G-protein-coupled receptors interact with heterotrimeric G-proteins upon activation. Receptor activation results in initiation of G-protein mediated signal transduction cascades leading to up- or down-regulation of intracellular levels of secondary messengers such as cAMP, inositol trisphosphate, or Ca2+. Termination of the receptor-mediated signaling is regulated by second messenger+regulated kinases and G-protein-coupled receptor kinases (GRKs). GRK-mediated phosphorylation of the receptor promotes receptor recruitment of the intracellular scaffolding proteins βarrestins (βarrs) from the cytosol, thereby excluding the receptor from further G-protein interaction (Luttrell and Lefkowitz, 2002). In addition to uncoupling 7TM receptors from G-proteins, βarrs recruit the receptor to clathrin-coated pits (Goodman et al., 1996) and initiates signaling pathways such as mitogen-activated protein kinase cascades (Luttrell and Lefkowitz, 2002). Seven members of the mammalian GRK family have so far been described. These GRKs are classified in three subgroups based on sequence homology and function: the visual GRK1 and -7, nonvisual cytosolic GRK2 and -3, and nonvisual membrane-associated GRKs 4, 5, and 6. With GRK4 as the exception, nonvisual GRKs are rather ubiquitously expressed in mammalian tissues. Although very little is known about GRK subtype-specific phosphorylation patterns of 7TM receptors, recent publications report GRK subtype-specific initiation of cellular events (Kim et al., 2005; Ren et al., 2005). At present, it is unknown how GRK subtype-specific functional effects are obtained, and details of the interaction pattern between 7TM receptors and GRK subtypes remain largely undescribed. Protein-protein interactions have been analyzed using coimmunoprecipitation techniques. For more detailed analysis, coimmunoprecipitation has several limitations, including the necessity for lysis and detergent treatment of the cells as well as low time resolution, making it difficult to detect transient interactions. For the analysis of GRK2 and GRK5 interaction with the NK-1 receptor, we used the bioluminescence resonance energy transfer (BRET) technique because it offers a relatively high-resolution method for analysis of biological interactions in living cells (Perroy et al., 2004; Galés et al., 2005, 2006). The BRET technique has previously been employed in the analysis of GRK2 but not GRK5 interaction with 7TM receptors (Hasbi et al., 2004; Jorgensen et al., 2007).
Molecular Biology. Human βarr2 N-terminally tagged with green fluorescent protein2 (GFP2-βarr2) and the Renilla reniformis luciferase (RLuc) cDNA were purchased from PerkinElmer Life and Analytical Sciences (Waltham, MA). Human GRK2 and GRK5 cDNA were cloned from a pancreatic cDNA library (Clontech, Mountain View, CA). GFP2 or RLuc fusions were generated using standard methods. GRK2-GFP2-Membrane was made by inserting the cDNA sequence encoding the C-terminal 17 amino acid residues from human K-ras (KDGKKKKKKSKTKCVIMS) followed by a stop codon in a3' position of the GRK2-GFP2 cDNA lacking the stop codon. Mutations were made using QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). All cDNA clones were verified by sequencing. Substance P (SP) peptide was obtained from Bachem (Bubendorf, Switzerland).
Transfections and Tissue Culture. cDNAs were transiently expressed in HEK293 cells transfected by calcium phosphate precipitation according to methods reported previously (Elling and Schwartz, 1996). HEK293 cells were obtained from the European Collection of Animal Cell Cultures. The cells were routinely maintained and passaged as recommended by the supplier.
Inositol Phosphate Assay. Agonist induced inositol phosphate accumulation was measured as described previously (Liu et al., 2003).
BRET2 Assays. BRET2 measurement was done using a Mithras LB 940 plate reader (Berthold Technologies, Bad Wildbad, Germany) as described previously (Jorgensen et al., 2007). All measurements were made at room temperature. The reading time was 5 min after agonist addition for dose-response curves. For kinetic studies, variable reading times were used, and agonist was added with an injector. For BRET2 saturation experiments, a range of transfections were made with a stable amount of NK1R-RLuc cDNA and increasing amounts of cDNA encoding the GFP2 fused protein. Reading times were as indicated in the figures. The background signal from Rluc was determined by coexpressing the Rluc construct with empty vector, and the BRET2 ratio generated from this transfection was subtracted from all other BRET2 ratios. Expression levels of Rluc- and GFP2-tagged constructs for each BRET2 saturation experiment were monitored by luminescence and fluorescence measurements. Luminescence was measured on the Mithras LB 940 plate reader two seconds after Deep Blue C (Coelenterazine 400a; PerkinElmer Life and Analytical Sciences) addition. For fluorescence measurements, cells from the same transfections were plated in a black clear-bottom microplate (ViewPlate; PerkinElmer Life and Analytical Sciences) at a density of 5 x 104 cells per well. After 1-h incubation in darkness, the total fluorescence was measured using the NovoStar microplate reader (BMG LabTech, Offenburg, Germany), with an excitation line at 485 nm and an emission filter at 520 nm. Background values obtained with cells transfected with the relevant RLuc construct but no GFP2 construct were subtracted, and the means of quadruplicate wells/sample were then calculated.
Confocal Fluorescence Microscopy. HEK293T cells were grown to 50% confluence on poly-L-lysine-coated, 22-mm diameter glass coverslips and transiently transfected (Lipofectamine; Invitrogen, Paisley, UK) with the FLAG-NK1 receptor and GRK5-GFP2, GRK2-GFP2, or GRK2-GFP2-Membrane cDNAs (0.5 µg + 0.5 µg per well). After 24 h, the medium was exchanged for serum-free Opti-MEM (Invitrogen) including 1% bovine serum albumin (30 min, 37°C), and the cells were then incubated for 30 min at 37°C in Opti-MEM/1% bovine serum albumin including M2 anti FLAG antibody (2.5 µg/ml, 150 µl per coverslip; Sigma, Poole, UK). Vehicle (50 µl of medium) or SP (10 nM final concentration) were added for 5 min, and cells were then washed in phosphate-buffered saline (PBS), fixed in 3% paraformaldehyde in PBS (15 min, 21°C), and permeabilized with 0.075% Triton X-100 for 5 min. Bound M2 antibody was detected by goat anti-mouse IgG conjugated to Rhodamine Red X (1:1000; Invitrogen). Cells were postfixed in 3% paraformaldehyde, and coverslips were mounted in 1:1 PBS/glycerol, sealed with nail varnish. Confocal microscopy was performed on a Zeiss LSM 510M laser-scanning microscope with a Plan-Apochromat 63x 1.4 numerical aperture oil objective. Images were acquired in multitrack mode using an argon laser line at 488 nm and bandpass emission filter at 505 to 550 nm (GRK-GFP2), and helium-neon laser with excitation at 543 nm and a long-pass filter at 560 nm (Rhodamine Red X secondary antibody). Ten to 15 representative cells were scanned for each data group, using gain and offset adjustments to obtain a full range of image intensities without saturation. Channel acquisition parameters were similar in the control and SP-treated examples given in Fig. 5.
Fig. 5. Confocal microscopy in HEK293 cells coexpressing FLAG-NK-1 receptor and either GRK5-GFP2 (A-F) or GRK2-GFP2-Membrane (G-L). Visualization of GRK5-GFP2 (A and D) or FLAG-NK-1 receptor (B and E) in coexpressing cells that have been stimulated with either vehicle (A-C) or 10 nM SP (D-F) for 5 min at 37°C. C and F are overlays of A plus B and D plus E, respectively. Visualization of GRK2-GFP2-Membrane (G and J) or FLAG-NK-1 receptor (H and K) in coexpressing cells that have been stimulated with either vehicle (G-I) or 10 nM SP (J-L) for 5 min at 37°C. I and L are overlays of G plus H and J plus K, respectively. Panels to the right of C and I represent amplifications of the areas marked with a white box in the respective panels. White bars correspond to a 10-µm scale.
Calcium Measurements. Calcium measurements were made using the NovoStar microplate reader (BMG LabTech, Offenburg, Germany) as described previously (Kubale et al., 2007).
Receptor Internalization. NK-1 receptor internalization was measured using the acid wash method with 125I-SP tracer (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK) as described previously (Kubale et al., 2007).
Data Analysis. Nonlinear regression analysis was made using Prism (GraphPad Software, Inc., San Diego, CA). BRET50 values were determined by one-phase exponential association or two-phase exponential association using Prism.
The NK-1 receptor, a prototypical family A 7TM receptor, has been demonstrated to undergo GRK2- and GRK5-mediated phosphorylation in vitro (Nishimura et al., 1998; Walker et al., 1999). However, the interaction pattern of the receptor and the GRKs in living cells has not been described. To analyze the interactions between the NK-1 receptor and GRK2 and GRK5, respectively, we employed a BRET assay in transfected HEK293 cells. The BRET assay is a proximity based assay that measures energy transfer from an Rluc-tagged protein to a GFP-tagged protein upon addition of the Rluc substrate Deep Blue C (Mercier et al., 2002; Milligan, 2004). The BRET signal is highly distance-dependent and is obtained if the Rluc energy donor and the GFP acceptor are within approximately 100 Å of each other. We measured BRET2 by using the GFP2 blue-shifted variant of GFP. We tagged the NK-1 receptor C-terminally with RLuc (NK1R-RLuc) and the GRKs C-terminally with GFP2 (GRK2-GFP2 and GRK5-GFP2). Functional expression of the NK1R-RLuc construct has been demonstrated (Vrecl et al., 2004), and other reports use GRK2 and GRK5 with C-terminal tags (Schulz et al., 2002; Li et al., 2003; Hasbi et al., 2004; Thiyagarajan et al., 2004; Jorgensen et al., 2007).
Fig. 1. Agonist-induced inositol phosphate accumulation. Dose response curves for agonist-induced inositol phosphate accumulation in HEK293 cells transiently transfected with cDNA encoding the NK-1 receptor and the indicated cDNAs. A, SP induced inositol phosphate accumulation in cells with NK-1 receptor coexpressed with either vector, GRK2, GRK2-GFP2, GRK2(K220R), or GRK2(K220R)-GFP2. B, SP induced inositol phosphate accumulation in cells with NK-1 receptor coexpressed with either vector, GRK5, GRK5-GFP2, GRK5(K215R), or GRK5(K215R)-GFP2. C, SP induced inositol phosphate accumulation in cells with tail truncated NK-1(1-324) receptor coexpressed with either vector, GRK5, GRK5-GFP2, GRK2, or GRK2-GFP2. The response is expressed as percent of the maximal response for the receptor cotransfected with empty vector. The curve for NK-1R+vector in A and B is identical. Results are the means ± S.E.M. of duplicate determinations in three independent experiments.
To further validate the functionality of GFP2-tagged GRK constructs and to analyze the ability of GRKs to regulate NK-1 receptor signaling, we measured the ability of the NK-1 receptor to mediate agonist induced inositol phosphate accumulation after coexpression with either vector, GRK2, GRK2-GFP2, GRK2(K220R), GRK2(K220R)-GFP2, GRK5, GRK5-GFP2, GRK5(K215R), or GRK5(K215R)-GFP2. The GRK2(K220R) and GRK5(K215R) mutants have been demonstrated to be kinase-inactive and are in some reports described to be dominant-negative (Kong et al., 1994; Diviani et al., 1996; Pronin and Benovic, 1997; Tiruppathi et al., 2000). We observed that coexpression of the NK-1 receptor with either GRK2 construct caused a substantial attenuation of agonist-induced inositol phosphate accumulation (Fig. 1A), demonstrating that GRK2 can regulate NK-1 receptor signaling and that the GFP2 fused GRK2 is functional. Furthermore, the ability of the kinase-inactive GRK2 mutants to attenuate NK-1 receptor-mediated signaling indicates that GRK2 can exert a phosphorylation-independent effect on receptor signaling. When measuring receptor-mediated inositol phosphate accumulation in the presence of coexpressed GRK5 constructs, we found that both GRK5 and GRK5-GFP2 coexpression attenuated receptor signaling to a similar level, suggesting that GRK5 can regulate NK-1 receptor signaling and that GFP2-fused GRK5 remains functional (Fig. 1B). Coexpression of the kinase-inactive GRK5(K215) mutant seemed to cause a relatively modest attenuation of NK-1 receptor-mediated inositol phosphate accumulation (Fig. 1B). However, the effect was not significant (Student's t test) and was not observed with GFP2-fused GRK5(K215R) construct, suggesting that GRK5 does not exert phosphorylation-independent effects on NK-1 receptor signaling.
To further distinguish phosphorylation-dependent and -independent effects of GRK2 and GRK5 on NK-1 receptor signaling, we employed a naturally occurring splice variant of the NK-1 receptor in which all of the receptor tail C-terminal to the palmitoylated Cys is lacking (Richardson et al., 2003) The NK1R(1-324) variant (numbers referring to remaining amino acid residues) has been reported not to be phosphorylated and to have reduced ability to interact with βarrs (Oakley et al., 2001; Richardson et al., 2003). We measured the ability of the tail-truncated NK-1R(1-324) construct to mediate agonist induced inositol phosphate accumulation when coexpressed with either vector, GRK5, GRK5-GFP2, GRK2, or GRK2-GFP2 (Fig. 1C). Coexpression of GRK5 or GRK5-GFP2 did not influence the signaling of the NK-1R(1-324) construct, whereas GRK2 and GRK2-GFP2 coexpression attenuated NK-1R(1-324)-mediated inositol phosphate accumulation, although to a lesser degree than observed with the wild+type receptor [maximal signal with GRK2 coexpression relative to control was 2% and 28% for NK1R(wt) and NK1(1-324), respectively]. Thus, the data suggest that the GRK5 effect on NK-1 receptor signaling was dependent on intact GRK5 kinase activity and on the presence of the receptor C-terminal tail, whereas GRK2 seems to have both phosphorylation-dependent and -independent effects on NK-1 receptor signaling.
To ensure that the NK-1 receptor is not constitutively desensitized when coexpressed with GRK2 or GRK5, we analyzed whether the receptor could generate a calcium signal in the initial 50 s after agonist addition. As seen in Fig. 2 both wild-type NK-1 receptor and NK1R-RLuc coexpressed with either vector, GRK2, or GRK5 mediates a calcium signal immediately after agonist addition. Thus, GRK coexpression does not cause constitutive desensitization of the NK-1 receptor, although a suppression of the max signal is observed in the presence of GRK coexpression (Fig. 2). As the time-dependent decrease of the peak in agonist-induced calcium signal reflects receptor desensitization as well as postreceptor mechanisms, we normalized the signals to percentage of maximum signal for optimal comparison (Fig. 2, inset) (Kubale et al., 2007). Coexpression of GRK2 or GRK5 caused the calcium signal to decrease with faster kinetics relative to coexpression with vector, indicating that the receptor desensitizes faster when GRKs are coexpressed (Fig. 2, inset). Furthermore, the data suggest that the NK1R-RLuc fusion is comparable with NK-1R(wt) in the ability to generate an agonist-induced calcium signal (Fig. 2).
Fig. 2. Agonist-induced calcium signal. Kinetics of agonist (10 nM SP)-induced calcium signal in transiently transfected HEK293 cells. The cells were either transfected with cDNAs encoding NK1R(wt)+ vector or NK1R-RLuc cotransfected with vector, GRK2-GFP2, or GRK5-GFP2 as indicated (cDNA ratio of 1:2). The inserted figure shows the signals normalized to percent of maximal generated calcium signal for each transfection condition. Data represents the mean ± S.E.M. for a single experiment carried out in sextuplicate determinations and are representative of a total of three independent experiments.
We next assessed the interaction between NK1R-RLuc and GRK5-GFP2 in transfected HEK293 cells by analyzing the BRET2 signal at progressive time points after addition of 10 nM SP—an agonist concentration that we found to have full efficacy in inducing recruitment of βarr2 to the NK-1 receptor (Supplementary Fig. 1). When measuring BRET2 between NK1R-RLuc and GRK5-GFP2, we observed a relatively high basal signal that increased to reach a transient peak at 20 s after agonist addition and returned to basal after 1 min (Fig. 3A). To analyze the interaction between the NK-1 receptor and GRK5 in more detail, we performed BRET2 saturation experiments. In these experiments, the amount of the GFP2-fused protein is gradually increased relative to a steady amount of the RLuc-fused protein (Mercier et al., 2002; Galés et al., 2006). In case of a specific interaction between the tagged proteins, the BRET2 signal should display a hyperbolic curve as a function of the GFP2/RLuc expression ratio (Mercier et al., 2002; Galés et al., 2006). In contrast, nonspecific background arising from random collisions of the tagged proteins ("bystander BRET") should result in a BRET2 signal that depends linearly on the GFP2/RLuc expression ratio (Mercier et al., 2002; Galés et al., 2006). We observed a hyperbolic curve between GRK5-GFP2 and the NK1R-RLuc, indicating that there is a basal association of GRK5 with the resting (i.e., receptor before agonist exposure) NK-1 receptor (Fig. 3B). The GFP/RLuc ratio resulting in half-maximal saturation is referred to as the BRET50 value and is a measure of the affinity in arbitrary units (Mercier et al., 2002; Galés et al., 2006). Hence, BRET2 saturation experiments are suitable for comparing affinities on a relative scale. Twenty seconds of receptor stimulation with 10 nM SP (corresponding to the time point of the peak in the BRET2 signal between GRK5-GFP2 and NK1R-RLuc) caused the curve to saturate with a lower BRET50 value, reflecting increased affinity of GRK5 for the receptor (BRET50 values of 0.19 and 0.48 with and without agonist addition, respectively). Thus, the BRET2 saturation data are consistent with the time course experiments and demonstrate that GRK5 is recruited during receptor activation to form a closer complex with the NK-1 receptor 20 s after receptor activation. Because some random interaction will occur between any proteins it was important to control that the apparent specific interaction between GRK5 and the resting receptor did not simply arise from bystander BRET2 between two membrane-localized proteins. The tail-truncated NK-1(1-324), which, in the inositol phosphate signaling assay, was observed to be signaling competent but not regulated by GRK5, was C-terminally RLuc tagged for the experiments (NK1R(1-324)-RLuc). The NK1R(1-324)-RLuc construct mediated agonist induced inositol phosphate accumulation virtually similar to the full-length NK1R-RLuc, demonstrating also that the RLuc-fused tail-truncated receptor construct was expressed and signaling competent (Supplementary Fig. 2). When performing BRET2 saturation experiments with the truncated NK1R(1-324)-RLuc construct and GRK5-GFP2, we observed a linear curve as a function of the GFP2/RLuc ratio both before and after agonist addition, suggesting that the NK1R(1-324)-RLuc construct did not interact with GRK5 (Fig. 3B). Furthermore, this suggested that the BRET2 signal between NK1R-RLuc and GRK5-GFP2 was not simply caused by both proteins being localized to the membrane. We further proceeded to characterize the interaction pattern of the kinase inactive GRK5(K215R) mutant with the NK-1 receptor. We were surprised to find that when performing time-course experiments with NK1R-RLuc and GRK5(K215R)-GFP2, we observed no agonist-induced increase in the BRET2 signal but rather a time-dependent decrease in the signal, reaching a plateau 1 min after agonist addition (Fig. 3C). Furthermore, the basal BRET2 signal was higher than observed with the NK1R-RLuc/GRK5-GFP2 combination (Fig. 3). When performing BRET2 saturation experiments with the NK1R-RLuc and GRK5(K215R)-GFP2, we observed the BRET2 signal, without agonist addition, to describe a slightly hyperbolic curve as a function of the GRK5(K215R)-GFP2/NK1R-RLuc ratio (Fig. 3D). Compared with GRK5-GFP2 interaction with NK1R-RLuc, the basal BRET2 signal for GRK5(K215R)-GFP2 was higher over a range of expression ratios, but it did not reflect a higher affinity phenotype (Fig. 3, B and D). Twenty seconds after addition of the agonist SP, the maximum BRET2 signal decreased without concomitant changes in the shape of the curve (Fig. 3D). Thus, the BRET2 data suggest that the GRK5(K215R) mutant is organized in a low affinity association with the resting NK-1 receptor and that the mutant is not further recruited to the receptor after receptor activation.
Fig. 3. BRET2 measurements with the NK-1 receptor and GRK5 constructs. A, time course of GRK5-GFP2 interaction with NK1R-RLuc after the addition of 10 nM SP. B, BRET2 saturation experiments with increasing amounts of GRK5-GFP2 relative to either NK1R-RLuc or NK1R(1-324)-RLuc. Measurements were made without receptor stimulation (buffer) or stimulation with 10 nM SP for 20 s. C, time course of GRK5(K215R)-GFP2 interaction with NK1R-RLuc after the addition of 10 nM SP. D, BRET2 saturation experiments with increasing amounts of GRK5(K215R)-GFP2 relative to NK1R-RLuc. Measurements were made without receptor stimulation (buffer) or stimulation with 10 nM SP for 20 s. A and C, results are the means ± S.E.M. of duplicate determinations in at least four independent experiments. B and D, data are pooled from at least three independent experiments.
We proceeded by characterizing the interaction between the NK-1 receptor and GRK2. When performing BRET2 time course experiments with NK1R-RLuc and GRK2-GFP2 we observed a rapid recruitment of GRK2-GFP2 to the NK1R-RLuc (Fig. 4A). The signal peaked transiently at 10 to 20 s after agonist addition and then stabilized at a lower level (Fig. 4A). When performing BRET2 saturation experiments, the BRET2 signal after agonist addition described a hyperbolic curve as a function of GRK2-GFP2/NK1R-RLuc that was best fit with a two-phase exponential association model (BRET50 values of 0.015 and 0.17 for the high- and low-affinity phases, respectively, measured 5 min after agonist addition) (Fig. 4B). It is noteworthy that the curve after 20 s of agonist addition (corresponding to the peak in the BRET2 signal in the time course measurements) was similar to the curve after 5 min of agonist addition, suggesting that the transient peak does not describe a higher affinity phenotype of the GRK2/NK-1 receptor complex relative to the signal plateau (Fig. 4B). As a control, we performed BRET2 saturation experiments with GRK2-GFP2 and the receptor tail truncated NK1R(1-324)-RLuc. Without agonist addition, the BRET2 signal displayed a linear curve as a function of the GRK2-GFP2/NK1R(1-324)-RLuc (Fig. 4B). Twenty seconds of agonist addition caused a slight increase in BRET2 signal, which may suggest that there is a low level of agonist-dependent GRK2 recruitment to the NK1R(1-324) (Fig. 4B). To analyze the dependence of GRK2 kinase activity for the observed interaction with the NK-1 receptor, we performed BRET2 time-course experiments of NK1R-RLuc interaction with the kinase inactive GRK2 mutant GRK2(K220R)-GFP2 after addition of 10 nM SP. In contrast to the results obtained with GRK5, the kinase inactive GRK2(K220R) mutant was recruited to the activated NK-1 receptor in a pattern similar to the wild-type GRK2 (Fig. 4C). We proceeded by performing BRET2 saturation experiments with GRK2(K220R)-GFP2 and NK1R-RLuc. We observed GRK2(K220R) interaction with the NK-1 receptor to saturate in a monophasic manner with a BRET50 value of 0.019 corresponding to the high-affinity phase of GRK2 interaction with the NK-1 receptor (Fig. 4D). Thus, the biphasic nature of the BRET2 saturation curve for GRK2 interaction with the NK-1 receptor seemed to consist of kinase-independent and -dependent phases.
Fig. 4. BRET2 measurements with the NK-1 receptor and GRK2 constructs. A, time course of GRK2-GFP2 interaction with NK1R-RLuc after the addition of 10 nM SP. B, BRET2 saturation experiments with increasing amounts of GRK2-GFP2 relative to either NK1R-RLuc or NK1R(1-324)-RLuc. Measurements were made without receptor stimulation (buffer) or stimulation with 10 nM SP for 20 s or 5 min. C, time course of GRK2(K220R)-GFP2 interaction with NK1R-RLuc after the addition of 10 nM SP. D, BRET2 saturation experiments with increasing amounts of GRK2(K220R)-GFP2 relative to NK1R-RLuc. Measurements were made without receptor stimulation (buffer) or stimulation with 10 nM SP for 20 s. E, BRET2 saturation experiments with increasing amounts of GRK2-GFP2-Membrane relative to NK1R-RLuc. Measurements were made without receptor stimulation (buffer) or stimulation with 10 nM SP for 20 s. Inset in E shows amplification of the areas of the curve marked with a dotted box. A and C, results are the means ± S.E.M. of duplicate determinations in at least three independent experiments. B, D, and E, data are pooled from at least three independent experiments.
To analyze whether the difference in phenotype of GRK2 and GRK5 interaction with the NK-1 receptor depends on the difference in cellular localization of the two GRKs, we made a membrane localized GRK2-GFP2 (GRK2-GFP2-Membrane) by fusing the C-terminal 17 amino acid residues from human K-ras to the C terminus of GRK2-GFP2. This sequence contains a polybasic signal and a prenylation sequence that in combination ensure localization to the plasma membrane (Hancock et al., 1991). We observed coexpression with GRK2-GFP2-Membrane to suppress NK-1 receptor-mediated inositol phosphate accumulation to less than 1% of control, demonstrating that the construct was functional (data not shown). The BRET2 signal was observed to saturate in a monophasic manner as a function of GRK2-GFP2-Membrane/NK1R-RLuc (Fig. 4E). After agonist addition, the saturation curve was best fit with a two-phase exponential association model (p = 0.0005) with the additional high-affinity phase as the minor phase (Fig. 4E, inset). Thus, when GRK2 is artificially membrane-localized, it displays basal interaction with the NK-1 receptor, but some agonist-induced recruitment can still be observed.
Because the BRET2 data did not reveal where in the cell an interaction occured, we performed confocal microcopy with a FLAG-tagged NK-1 receptor and either GRK5-GFP2 or GRK2-GFP2 to see whether the proteins dissociate after receptor internalization. We observed the unstimulated NK-1 receptor to be localized to the plasma membrane in a clustered pattern (Fig. 5, B and C). As expected, GRK5-GFP2 was also membrane-localized, and some clustering was observed in a pattern overlapping NK-1 receptor localization (Fig. 5, A and C). After 5 min of stimulation with 10 nM SP, extensive NK-1 receptor internalization was observed without concomitant internalization of GRK5 (Fig. 5, D-F). Thus, GRK5 colocalizes with the NK-1 receptor in membrane clusters, but it does not cointernalize with the receptor. When FLAG-NK1R was coexpressed with GRK2-GFP2, we again observed extensive receptor internalization (Supplementary Fig. 3). Although GRK2-GFP2 did not seem to cointernalize with the receptor, the cytoplasmic localization of GRK2-GFP2 made it difficult to conclude on this issue (Supplementary Fig. 3). Therefore, we took advantage of the membrane localization of the GRK2-GFP2-Membrane construct to analyze the internalization pattern. We observed GRK2-GFP2-Membrane to be localized to the plasma membrane as expected (Fig. 5G). Although surface localization of FLAG-NK1R before agonist addition was still observed when coexpressed with GRK2-GFP2-Membrane, there was relatively more constitutively internalized receptor compared with coexpression with GRK5-GFP2 or GRK2-GFP2 (compare Fig. 5, B and H, and supplementary Fig. 3B). After 5 min of agonist exposure, the receptor internalized without any apparent concomitant internalization of GRK2-GFP2-Membrane (Fig. 5, J-L). Hence, GRK2-GFP2-Membrane seems to increase constitutive internalization of the NK-1 but does not cointernalize with the receptor.
Fig. 6. Effect on BRET2 signals of inhibition of receptor internalization by hypertonic sucrose. A, time course of GRK5-GFP2 interaction with NK1R-RLuc after the addition of 10 nM SP with or without addition of 450 mM sucrose to the assay buffer as indicated. B, time course of GRK2-GFP2 interaction with NK1R-RLuc after the addition of 10 nM SP with or without addition of 450 mM sucrose to the assay buffer as indicated. Results are the means ± S.E.M. of duplicate determinations in three independent experiments.
To analyze the influence of receptor internalization on the observed pattern of GRK2 and GRK5 interaction with the NK-1 receptor, we performed BRET2 time course experiments in the presence 450 mM sucrose to inhibit receptor internalization (Heuser and Anderson, 1989). The BRET2 assay was not affected by addition of sucrose to the assay buffer (data not shown). In a control experiment, we found 450 mM sucrose to significantly inhibit the kinetics of agonist-induced NK-1 receptor internalization and to inhibit the maximal internalization (Supplementary Fig. 4). Five minutes after agonist addition, hypertonic sucrose caused 65% inhibition of receptor internalization relative to control (Supplementary Fig. 4). Both when measuring GRK5-GFP2 and GRK2-GFP2 recruitment to NK1R-RLuc (Fig. 6, A and B, respectively), we found the addition of sucrose in the buffer to cause a considerable increase in the basal BRET2 signal. However, the pattern of the agonist-induced BRET2 signal as a function of time did not differ between the experiments with or without addition of sucrose to the buffer. Thus, the observed decrease of the peak BRET2 signal between the NK-1 receptor and GRK5 or GRK2 in time-course experiments seems to be independent of receptor internalization.
Because it is unknown whether GRK subtypes compete directly for the receptor substrate, we performed BRET2 competition experiments. In these experiments, an untagged protein is coexpressed with the pair of tagged proteins to analyze for competition (Marullo and Bouvier, 2007). To analyze for competition of GRK5 recruitment to the NK-1 receptor, we coexpressed NK1R-RLuc with GRK5-GFP2 and either vector, GRK2(wt), GRK2(K220R), GRK5(wt), or GRK5(K215R) (Fig. 7A). It is noteworthy that only GRK5(wt) competed for agonist-induced GRK5-GFP2 interaction with NK1R-RLuc (Fig. 7A). The ratio of GFP2/RLuc did not significantly differ between the transfection conditions and was within the saturated range (range of GFP2/RLuc was 0.61-0.86) (Supplementary Fig. 5A). When analyzing the ability of untagged proteins to compete for agonist-induced recruitment of GRK2-GFP2 to NK1R-RLuc, we found both GRK2(wt), GRK2(K220R), GRK5(wt), and GRK5(K215R) to compete (Fig. 7B). Compared with GRK2(wt), GRK2(K220R), and GRK5(wt), untagged GRK5(K215R) was far less efficacious in the ability to compete for agonist-induced recruitment of GRK2-GFP2 to NK1R-RLuc, and the competition only reached significance at time points 20 and 45 s after agonist addition (Fig. 7B). The range of GFP2/RLuc was 0.83-1.39 (plus vector = 1.1) and thus within the saturated area (Supplementary Fig. 5B).
Fig. 7. BRET2 competition experiments. Time course of GRK5-GFP2 (A) or GRK2-GFP2 (B) interaction with NK1R-RLuc after the addition of 10 nM SP. The cells were cotransfected with the indicated cDNAs to analyze for the ability to compete with the GFP2-fused GRKs for NK1R-Luc interaction. Results are the means ± S.E.M. of duplicate determinations in five (A) or four (B) independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus values for cotransfection with vector (unpaired Student's t test).
We here characterize the interaction pattern of GRK2 and GRK5 with the NK-1 receptor. Although the role of GRKs in phosphorylating activated 7TM receptors, thereby promoting binding of βarrs, is well established (Luttrell and Lefkowitz, 2002; Pao and Benovic, 2002), details of the interaction pattern of GRK subtypes with a 7TM receptor have never been compared.
In the analysis of GRK effect on NK-1 receptor inositol phosphate signaling, we observed both GRK2 and GRK5 to attenuate NK-1 receptor signaling. It is noteworthy that GRK2 seemed to have a kinase-independent effect on NK-1 receptor signaling. In contrast, the observed effect of GRK5 on NK-1 receptor signaling seemed to be kinase-dependent. A kinase-independent effect of GRK2 is not unprecedented in that GRK2 and GRK3, in several instances, have been reported to exert phosphorylation-independent roles in 7TM receptor desensitization (Pao and Benovic, 2002). GRK2 and -3 (but not GRK5) can bind G-protein Gq/11 and Gβ subunits, and the phosphorylation-independent effect on receptor desensitization is thought to originate from sequestering of these G-protein subunits and stimulation of G-protein GTPase activity (Pitcher et al., 1992; Kunapuli and Benovic, 1993; Carman et al., 1999b; Pao and Benovic, 2002). In fact, GRK2 and -3 are reported to depend on interaction with liberated Gβ subunits for recruitment to activated 7TM receptors (Pitcher et al., 1992). In a BRET2 assay, we observed limited agonist-induced interaction between a tail truncated NK-1R(1-324) construct and GRK2, which indicates that the effect of GRK2 on G-protein signaling of this receptor construct is not based on receptor interaction per se. This is consistent with the paradigm that kinase-independent effects of GRK2 are mediated by interaction with G-protein subunits rather than direct modulation of the receptor. Furthermore, the results suggest that the NK-1 receptor C-terminal tail is necessary for quantitative GRK2 interaction.
When analyzing GRK interaction with the NK-1 receptor, we observed several interesting differences in the GRK subtype-specific interaction patterns. Agonist-induced interaction of GRK5 with the NK-1 receptor was kinase-dependent and saturated in a monophasic manner, whereas GRK2 saturated in a biphasic manner, with the high-affinity phase being independent of kinase activity. It is possible that the high-affinity phase of GRK2 interaction with the activated NK-1 receptor represents initial GRK2 recruitment, whereas the lower affinity phase represents a conformational change promoted by phosphorylation. It is interesting to note that the monophasic interaction of GRK5 with the activated receptor seems to correspond to the kinase-dependent phase of GRK2 interaction with the receptor (BRET50 values of 0.19 and 0.17, respectively). Thus, our data suggest a difference in recruitment modes for GRK2 and GRK5 to the activated NK-1 receptor.
BRET2 saturation experiments showed the basal BRET2 signal for NK-1 receptor interaction to be higher, at the same expression ratios, for GRK5(K215R) compared with GRK5. Because inhibition of receptor internalization with hypertonic sucrose was observed to increase the basal BRET2 signal, it is possible that GRK5(wt), but not GRK5(K215R), promotes constitutive phosphorylation with subsequent receptor internalization for a subpopulation of receptors, thereby decreasing the basal GRK5/NK-1 receptor BRET2 signal. The high basal BRET2 signal declined after receptor activation, indicating that GRK5(K215R), like GRK5(wt), is localized near the resting receptor and that dissociation/removal occurs after activation. The lack of agonist-induced receptor recruitment of GRK5(K215R) was surprising and clearly distinguishes it from the kinase-inactive GRK2-(K220R) mutant. However, in saturation experiments, GRK5 lacks the high-affinity phase of receptor interaction that is seen for GRK2 and retained in GRK2(K220R).
Our BRET2 saturation data indicate that GRK5 is preassociated with the resting NK-1 receptor and that receptor activation causes recruitment of GRK5 to a higher affinity complex with the receptor. The results do not allow us to distinguish between true translocation of GRK5 to the activated receptor from a nearby position (in, for example, a microdomain structure) and activation-dependent reorganization of a pre-existing GRK5/NK-1R complex. However, the BRET2 sensitive range of 100 Å corresponds to the diameter of only two to three 7TM receptors in the membrane plane, suggesting relatively close proximity (Palczewski et al., 2000). The notion that GRK5 may associate with resting 7TM receptors is further supported by reports that describe that overexpression of GRK5 but not GRK2 can increase basal receptor phosphorylation (Premont et al., 1994; Diviani et al., 1996; Shetzline et al., 1998, 2002). In addition, GRK5 sedimentation experiments with phospholipids versus rhodopsin-containing membranes have indicated that GRK5 can bind directly to receptors in the membrane (Pronin and Benovic, 1997). When analyzing the interaction between the NK-1 receptor and a membrane-localized GRK2 construct, we also observed an agonist-independent monophasic hyperbolic saturation curve, suggesting basal interaction. This indicates that the ability to preassociate with the NK-1 receptor depends on subcellular localization rather than properties inherent to specific GRK subtypes. However, several differences in the phenotype of GRK5 and membrane-attached GRK2 was observed. After receptor activation, the interaction of the NK-1 receptor with GRK2-GFP2-Membrane was best fitted with a two-phase association model that corresponds to the phenotype of GRK2. Furthermore, coexpression of the NK-1 receptor and GRK2-GFP2-Membrane was observed in confocal microscopy experiments to cause an increase in constitutive receptor internalization that was not as apparent with GRK2-GFP2 or GRK5-GFP2 coexpression, indicating that GRK2-GFP2-Membrane have constitutive effects on the NK-1 receptor.
When performing confocal microscopy, we observed colocalization of GRK5 and the resting NK-1 receptor to the plasma membrane (including some clusters), and GRK5 did not cointernalize with the NK-1 receptor, thereby demonstrating that the proteins dissociate before or during receptor internalization. Likewise, we observed no cointernalization of GRK2 (or membrane-associated GRK2) and the NK-1 receptor, suggesting that the plateau in BRET2 signal, observed in time-course experiments for GRK2 interaction with the NK-1 receptor, reflects a steady-state rather than quantitative cointernalization. After receptor internalization into cytoplasmic vesicles, increased random interaction with the cytosolic GRK2 may occur that would cause the background BRET2 signal to increase. Inhibition of receptor internalization with hypertonic sucrose did not prevent or delay the time-dependent decrease in BRET2 signals for agonist-dependent receptor interaction with either GRK2 or GRK5, suggesting that dissociation of GRK/NK-1R complexes occurs before receptor internalization.
BRET2 competition experiments suggested that GRK5 can compete for agonist-induced GRK2 recruitment to the NK-1 receptor, whereas GRK2 does not compete for GRK5 recruitment to the activated receptor. This demonstrates that GRK subtypes do compete for the receptor substrate and suggests that the competition is orchestrated by more than simply relative amounts of GRK subtypes or relative affinities for the activated receptor. Indeed, the competition data would support a hypothesis that GRK5 preassociation with the NK-1 receptor, causing a local high concentration of GRK5, gives an advantage of proximity that outweighs the higher affinity of GRK2 for the activated receptor. The competition between GRK subtypes could be both steric and functional. Steric competition between large proteins that interact with the same domain of the receptor would be expected, and GRK5-mediated receptor phosphorylation and subsequent uncoupling would prevent further GRK2 recruitment because GRK2 depends on G-protein activation for translocation (Pitcher et al., 1992).
The untagged GRK2(K220R) mutant was observed to compete for GRK2-GFP2 recruitment to the NK-1 receptor, thereby further supporting reports that the GRK2(K220R) mutant is dominant-negative (Kong et al., 1994; Diviani et al., 1996). However, as untagged GRK5(K215R), did not compete for GRK5-GFP2 recruitment to the activated NK-1 receptor, our results do not support the use of the GRK5-(K215R) mutant as a dominant-negative tool. It remains possible that GRK5(K215) has a competitive effect on basal GRK5 preassociation with the resting receptor that we do not see in our assay. Because the GRK constructs may have an effect on constitutive receptor internalization that the basal BRET2 signal seems to be highly sensitive to, we cannot determine whether effects of overexpressed untagged GRKs on the basal BRET2 signal reflects competition or changes in the ratio of surface receptor/total receptor.
Our data suggest that GRK5 rather than GRK2 is a key player in the regulation of GRK subtype-specific interaction with the NK-1 receptor. Because the ability of GRK5 to regulate GRK2 interaction with the receptor seems to be largely dependent on kinase activity, one could speculate that inhibition of GRK5 kinase activity would not only prevent GRK5-mediated receptor phosphorylation but, furthermore, would allow GRK2-mediated phosphorylation. In this context, it is interesting to note that GRK5 kinase activity can be inhibited by calcium/calmodulin and structural proteins such as actin, -actinin, and caveolin (Pronin et al., 1997; Freeman et al., 1998; Carman et al., 1999a; Freeman et al., 2000).
In conclusion, we demonstrate differences in affinity, kinase dependence, and level of basal interaction for GRK2 and GRK5 interaction with the NK-1 receptor. We suggest that, rather than relative amounts of GRK subtypes, GRK5 pre-association with the receptor is involved in the orchestration of subtype-specific GRK regulation of receptor signaling. It will be interesting to see future research delineate in more detail how the complex interaction pattern between receptors and GRKs, as well as other accessory proteins, is biologically regulated and how this contributes to the regulation of receptor signaling.
Acknowledgements
We thank Susanne Hummelgaard, Kate Hansen, Helle Zancho Andresen, and Helle Iversen for excellent technical assistance.
ABBREVIATIONS: 7TM, seven transmembrane; NK-1, neurokinin-1; GRK, G-protein-coupled receptor kinase; βarr, βarrestin; BRET, bioluminescence resonance energy transfer; RLuc, Renilla reniformis luciferase; SP, substance P; HEK, human embryonic kidney; BSA, bovine serum albumin; PBS, phosphate-buffered saline; wt, wild type.
The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
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作者单位：7TM Pharma A/S, Horsholm, Denmark (R.J., A.H., T.W.S., C.E.E.); Institute of Cell Signaling, Queen's Medical Centre, Nottingham, United Kingdom (N.D.H.); Institute of Anatomy, Histology, and Embryology, Veterinary Faculty, University of Ljubljana, Slovenia (M.V); Laboratory for Molecular Pharmacolog