Raf kinase inhibitory protein reduces bradykinin receptor desensitization

Abstract Inflammatory hyperalgesia represents a nociceptive phenotype that can become persistent in nature through dynamic protein modifications. However, a large gap in knowledge exists concerning how the integration of intracellular signaling molecules coordinates a persistent inflammatory phenotype. Herein, we demonstrate that Raf Kinase Anchoring Protein (RKIP) interrupts a vital canonical desensitization pathway to maintain bradykinin (BK) receptor activation in primary afferent neurons. Biochemical analyses of primary neuronal cultures indicate bradykinin‐stimulated PKC phosphorylation of RKIP at Ser153. Furthermore, BK exposure increases G‐protein Receptor Kinase 2 (GRK2) binding to RKIP, inhibiting pharmacological desensitization of the BK receptor. Additional studies found that molecular RKIP down‐regulation increases BK receptor desensitization in real‐time imaging of primary afferent neurons, identifying a key pathway integrator in the desensitization process that controls multiple GRK2‐sensitive G‐protein coupled receptors. Therefore, RKIP serves as an integral scaffolding protein that inhibits BK receptor desensitization.


| INTRODUC TI ON
Inflammatory hyperalgesia following tissue injury occurs as afferent nerve fibers become sensitized to physical and chemical stimuli.
Inflammatory mediators such as bradykinin (BK) activate receptors on afferent nerve endings (Dray, 1995) to reduce the activation threshold.
Despite rapid canonical desensitization mechanisms for G-protein coupled receptors (GPCRs) such as the bradykinin type-2 receptor (B2R) (Leeb-Lundberg et al., 2005), patients often experience persistent inflammatory sensitization following acute injury. This phenomenon has been understudied and is an important therapeutic target for patients experiencing persistent pain syndromes. The research herein identifies a scaffolding molecule that prolongs bradykinin receptor desensitization in primary afferent neurons and may serve some clinical utility.

G-protein Receptor Kinase (GRK) is a multi-domain enzyme ex-
pressed throughout mammalian tissues and is critically involved in G-protein association and GPCR desensitization. Several isoforms, including GRKs 2, 3, 5, and 6, phosphorylate GPCRS to stimulate βarrestin association and receptor internalization (Kelly et al., 2008).
RKIP serves as a multi-function scaffolding protein capable of serving as a "sink" for free cytosolic GRK2. RKIP phosphorylation at Ser153 by Protein Kinase C stimulates RKIP dimerization that can then bind to GRK2 and remove it from targeting other substrates, including GPCRs (Deiss et al., 2012;Lorenz et al., 2003). We hypothesized that a similar scenario could inhibit B2R desensitization in a more physiologically-relevant model. Experimental results herein indicate that RKIP binding to GRK-2 actively dictates B2R desensitization in primary afferent neuronal cultures, potentially contributing to the persistent nature of clinical inflammatory pain.

| Animals and materials
Procedures utilizing animals were approved by the University of Texas Health San Antonio's (UTHSA) Institutional Animal Care and Use Committee (IACUC, protocol 20130050AR, approved 04/20).
Studies were conducted in accordance with the policies for the ethical treatment of animals established by the National Institutes of Health (NIH) with every effort made to limit animal discomfort and the number of animals used. Sprague Dawley rats (3-4 weeks of age, 50-75 g; Charles River Laboratories, RRID:RGD_10395233) were used throughout this study. Animals were housed in clean, Allentown Static cages, 3/cage, with a 12 h light/dark cycle with food and water ad libitum before use. Unless otherwise stated, common chemical reagents were purchased through Sigma-Aldrich. were then re-suspended in complete media and plated on polydlysine/laminin-coated coverslips (BD Biosciences, 6 coverslips/rat [2 TGs or 6 DRGs], Cat# 354087 Cultures were maintained at 37°C and 5% CO 2 and grown for 1-2 days for functional studies or 5-6 days for biochemistry. Media was changed the day following initial culturing and every 2 days thereafter. TG were utilized for biochemical experiments to satisfy NIH requirements to reduce animal use in research.

| Immunohistochemistry and immunocytochemistry
Male Sprague-Dawley rats were anesthetized with an intraperitoneal (i.p.) injection of pentobarbital (100 mg/kg) and were transcardially perfused with 100 ml 0.9% saline in water followed by 250 ml Processed slides were dried and stored at −20°C. Cultured rat TG neurons were grown on poly-D lysine coated coverslips for 2-3 days in normal media. Coverslips were rinsed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde in 0.1 M PBS for 10 min at 25°C. Following fixation, coverslips were rinsed twice with PBS and incubated with 5% normal goat serum (Gibco, Cat# PCN5000, 0.5% Triton X-100 in PBS for 30 min at 25°C. Staining steps were performed at 22°C. Slides were rinsed in 0.1 M PBS three times for 10 min each. Samples were then incubated for 90 min in blocking solution (2% bovine -globulin (Sigma-Aldrich, Cat# 345876), 4% normal horse serum (Sigma-Aldrich, Cat# H0146), and 0.3% Triton X-100 (Fisher Scientific) in 0.1 M PBS) to minimize non-specific binding. Next, slides and coverslips were incubated in primary antibody against RKIP (Cat# 07-137, Millipore; at 1:100 dilution in blocking solution) for 18 h in a humidifier. Antibody specificity was validated with no immunoreactivity in RKIP knockout mice (Subramanian et al., 2014). Slides and coverslips were also incubated with a primary antibody specific for B2R (Clone 20/B2, BD Transduction Laboratories, at 1:100 dilution in blocking solution) and TOPRO and DAPI, as indicated and performed previously (Duchene et al., 2002).
Next, tissue was rinsed with 0.1 M PBS and placed in a dark humidifier for 90 min to incubate in anti-rabbit secondary-linked Alexa Fluor 488 (Molecular Probes at 1:100 dilution in blocking solution, to visualize RKIP immunoreactivity) and anti-mouse secondarylinked Alexa Fluor 594 (Molecular Probes at 1:100 dilution in blocking solution, to visualize B2R immunoreactivity). Nuclei marker TOPRO (Invitrogen; 1:5000 dilution) or DAPI (ThermoFisher; 1:5000 dilution) were added to the secondary antibody-blocking solution. The tissue was rinsed in 0.1 M PBS, followed by a final wash in deionized water. Once dry, stained DRG slides were coverslipped with Vectashield (Vector Labs), and stored at 4°C until they were evaluated by confocal microscopy. Coverslips were attached to Superfrost Plus slides, sealed, and stored at −20°C until confocal evaluation. Images were obtained using a Nikon 90i microscope equipped with a C1si laser scanning confocal imaging system.
Confocal images are representative of four individual trials.

| TG harvesting and Co-Immunoprecipitation (Co-IP)
On identified days and following specific treatments, cultures were rinsed twice with ice-cold PBS, brought down in Co-IP buffer (20 mM HEPES, 120 mM NaCL, 20 mM NaF, 20 mM 2-glycerol phosphate, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM orthovanadate, 0.1% Triton X-100, 1 mM benzamidine, pH to 7.45), lysed by 20 passes through a 25 g needle, and incubated on ice 15 min prior to clearing by centrifugation (500 μg, 1 min) and subsequent Bradford (Bradford, 1976) analysis of remaining supernatant for protein concentrations. Equal amounts of protein (500 μg) were immunoprecipitated with 1 μg anti-GRK2 (clone C-15, Santa Cruz Biotechnology) with GRK2 antibody specificity previously confirmed in inducible GRK2 sensory neuron knockout animals (Wang et al., 2011). 50 μg samples were kept for whole cell lysate analysis and confirmation of targeted protein expression. 500 μg samples were incubated with GRK2 Ab 18 h at 4°C on a shaking rack set to 180 rpm. Protein-A agarose beads (Abcam, Cat# ab193254) were incubated with 56°Ctreated bovine serum albumin for the same time period under similar conditions. Following primary antibody incubation, samples and beads were allowed to precipitate by gravity on ice, upon which samples and beads were combined and incubated at 4°C shaking for 2 h. Following this, samples were rinsed 4 times with Co-IP buffer and combined with SDS-PAGE loading buffer for WB analysis.

| Western blot (WB) analysis
Cleared TG lysate and Co-IP samples were resolved by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinyldifluoride membranes (PVDF; Millipore).
Integrated density measurement values, equivalent to the product of area and mean gray value by histogram analysis, were performed using NIH ImageJ software.

| PKC kinase activity
PKC activity was assessed in TG neurons treated with BK or PDBu (as indicated). Immediately following treatment, TG were collected in ice-cold PBS, washed twice with PBS, and suspended in 1X sample preparation buffer (50-mM Tris-HCl, 10 mM benzamidine, 5 mM EDTA, 10 mM EGTA, pH 7.5). Samples were sonicated five times for 10 s each and centrifuged at 100 000 g for 60 min at 4°C. The "Independent trial" corresponds to an independent culturing event, whereas samples were quantified in three equal aliquots ('triplicate').
The treatment timeline proceeded as follows: 1 min baseline, 30-sec BK administration, 5-min washout, 30-sec BK administration, 2-min washout. F340/380 was recorded in 3-sec bins. CAP and KCl sensitivity were determined after BK responses were recorded, with CAP(−) and/or KCl(−) cells removed from study inclusion. Results represent various n (typically >60 neurons) from >5 coverslips per BK concentration, across TGs from four rats bilaterally harvested and cultured for coverslip use. Positive transfection with RKIP siRNA was determined by FITC(+) expression in neurons. Homogenates were placed on ice for 15 min incubation and then centrifuged at 1000 g for 1 min to remove nuclei and unlysed cells from the homogenate. The resulting supernatant, whole-cell lysates, were collected and analyzed by WB.

| RE SULTS
Few studies have focused on protein expression patterns of RKIP in peripheral sensory neurons. Therefore, we performed immunocytochemical and immunohistochemical analyses of RKIP and B2R expression in cultured TG and intact DRG neurons. Both neuronal types were studied to determine potential differential expression patterns (Lindquist et al., 2021). In Figure 1, TG neuronal staining identifies strong RKIP (green, Figure 1a) co-expression with B2R (red, Figure 1b) in a heterogenous primary TG cultures in vitro (Figure 1d).
Similarly, RKIP (green, Figure 1e) and B2R (red, Figure 1f) demonstrate strong co-expression in intact DRG in vivo (Figure 1h). However, anatomical co-expression does not indicate functional RKIP modulation of BK receptor activity sensory neurons, which is necessary to establish translational relevance to feed-forward regulation.
As demonstrated in Figure 3a, TG cultures were treated with vehicle, 50 nM BK, BK + HOE-140 (1 μM, B2R antagonist) or BK + GFX (GF109203X, 10 μM, broad-spectrum PKC inhibitor). Physiological BK (50 nM) increased RKIP phosphorylation twofold in a manner sensitive to B2R antagonism and PKC inhibition. We also performed co-IPs from cultured sensory neurons treated with physiological BK to investigate RKIP association with GRK2. In Figure 3b, BK (50 nM and 1 μM) increased RKIP Co-IP with GRK2 compared to vehicletreated cultures. Interestingly, more concentrated BK treatment did not increase GRK2 association with RKIP as it did with increasing PKC activity (Figure 2). Also, acute treatment with BK failed to change the endogenous expression of GRK2. This suggests that at a physiological BK, far lower than what is used in many studies, RKIP maximally sequesters GRK-2 in primary sensory neurons.

| DISCUSS ION
Inflammatory mediators such as BK sensitize primary afferent sensory neurons to nociceptive stimuli via GPCR activation. In most in vitro models, GPCR activation stimulates a canonical internalization event that limits persistent activity. However, persistent inflammatory nociception remains unexplained. Herein, we report that RKIP drives a feed-forward regulatory system interrupting canonical B2R desensitization to maintain receptor responsivity. As illustrated in Figure 7, BK activation of B2R stimulates Gαq/11-driven PKC phosphorylation of RKIP at Ser153 to sequester GRK2 and prevent GRK2dependent facilitation of agonist-dependent B2R desensitization.
In an equimolar system, downstream signaling would be considered concentration-dependent throughout, as evidenced by PKC activity assay results in Figure 2. However, Ca +2 imaging results in Figure 4 indicate that secondary signaling pathways demonstrate differential variability in pharmacologic desensitization patterns.
Future studies should include physiological concentrations of BK lower than 50 nM, which would be expected to provide even greater sensitization in the absence of RKIP. However, it cannot be presumed that all downstream and regulatory signaling components exist equimolar to receptors, thereby influencing the interpretation that receptor occupancy directly dictates receptor desensitization.
Furthermore, the limitations of in vitro experimentation prevent us from modeling tachyphylactic agonist exposure as it likely exists in vivo, as a constant, non-pulsatile exposure. Nevertheless, our approach highlights an important regulator of GPCR responsivity in a manner unexplained by transfected cell models and supraphysiological agonists.
Supraphysiological treatment with BK (1-100 μM) rapidly desensitizes B2R in transfected (Blaukat et al., 1996;Fathy et al., 1999) and immortalized cell models (Smith et al., 1995;Wolsing & Rosenbaum, 1991) and is supported by our own observations ( Figure 4). However, this range of concentrations neither models human inflammation (Hargreaves et al., 1988a;Hargreaves & Costello, 1990) nor supports behavioral desensitization to acute nanomolar BK injections (Adachi & Ishii, 1979;Miao et al., 1996;Tonussi & Ferreira, 1997). However, a limitation of this study is the lack of consequent data on Store-Operated Calcium Entry (SOCE), such that following ER depletion of internal calcium stores, BK may induce calcium influx from the extracellular space. Indeed, BKstimulation of Orai1 produces transient increases in intracellular Ca +2 in sensory neurons (Szteyn et al., 2015), which could inflate outcome measures and confound direct interpretation. Another limitation includes the stability of BK in solution, given its sensitivity to degradation by peptidases including EP24.15 and EP24.16 expressed by sensory neurons (Jeske et al., 2006). Experiments reported here did not utilize peptidase inhibitors throughout Ca +2 imaging, resulting in under-reporting apparent BK concentrations. Future studies will account for these unintentionally biases and contribute more to the physiological relevance of this signaling phenomenon.
A number of GPCRs are negatively modulated by GRK2, supporting the possibility that RKIP sequestration of GRK2 could affect other receptor systems. Recent research identifies roles for RKIP in multiple physiologies (Granovsky et al., 2009), including cancer (Dangi-Garimella et al., 2009), inflammation (Wright & Vella, 2013), and cardiovascular function (Lorenz et al., 2003). Furthermore, tissue-specific differences in GRK2/GPCR interactions, such as constitutive DOR/MOR association with GRK2 sensory neurons (Brackley et al., 2016;Zhang & Jeske, 2020) could increase RKIP F I G U R E 5 Sirna knockdown of RKIP protein expression. Cultured TG sensory neurons transfected in mock fashion (Hiperfectamine only), with FITC(−) (FITC-labeled Ambion negative control), RKIP1 siRNA (designed against RKIP), or FITC-RKIP1 (same sequence as RKIP1, labeled with FITC for identification). Representative Western blot of cleared lysates probed for RKIP and β-Actin (loading control). Mean band densitometries ± SEM shown, **p < 0.01 compared to control, one-way ANOVA with Bonferroni correction, n = 3 independent primary culture preparations/treatment (p = 0.0007, F = 0.5070, DFn = 3) control of peripheral opioid analgesic efficacy. However, GRK2 interactions with non-GPCR proteins, including Akt and p38 (Penela et al., 2010) complicate experimental interpretations, especially in terms of disease models and translational relevance. Taken together, the results presented here identify a role for RKIP inhibition of BK receptor desensitization in sensory neurons and suggests a contributory role toward persistent inflammatory hyperalgesia.
Future studies should focus on other GPCRs affected by RKIP to explore the centralized utility of RKIP scaffolding and sequestration of GRK2.

ACK N OWLED G M ENTS
This work was supported by US National Institutes of Health (NIH) grants NS082746 and NS120276 (NAJ), and DE025551 (ADB).
All experiments were conducted in compliance with the ARRIVE guidelines.