Abstract
Upon agonist stimulation, α1B-adrenergic receptors couple to Gq proteins, calcium signaling and protein kinase C activation; subsequently, the receptors are phosphorylated, desensitized, and internalized. Internalization seems to involve scaffolding proteins, such as β-arrestin and clathrin. However, the fine mechanisms that participate remain unsolved. The roles of protein kinase C and the small GTPase, Rab9, in α1B-AR vesicular traffic were investigated by studying α1B-adrenergic receptor–Rab protein interactions, using Förster resonance energy transfer (FRET), confocal microscopy, and intracellular calcium quantitation. In human embryonic kidney 293 cells overexpressing Discosoma spp. red fluorescent protein (DsRed)-tagged α1B-ARs and enhanced green fluorescent protein–-tagged Rab proteins, pharmacological protein kinase C activation mimicked α1B-AR traffic elicited by nonrelated agents, such as sphingosine 1-phosphate (i.e., transient α1B-AR-Rab5 FRET signal followed by a sustained α1B-AR-Rab9 interaction), suggesting brief receptor localization in early endosomes and transfer to late endosomes. This latter interaction was abrogated by blocking protein kinase C activity, resulting in receptor retention at the plasma membrane. Similar effects were observed when a dominant-negative Rab9 mutant (Rab9-GDP) was employed. When α1B-adrenergic receptors that had been mutated at protein kinase C phosphorylation sites (S396A, S402A) were used, phorbol ester-induced desensitization of the calcium response was markedly decreased; however, interaction with Rab9 was only partially decreased and internalization was observed in response to phorbol esters and sphingosine 1-phosphate. Finally, Rab9-GDP expression did not affect adrenergic-mediated calcium response but abolished receptor traffic and altered desensitization. Data suggest that protein kinase C modulates α1B-adrenergic receptor transfer to late endosomes and that Rab9 regulates this process and participates in G protein-mediated signaling turn-off.
Introduction
α1B-Adrenergic receptors (α1B-ARs) are seven transmembrane-domain proteins that mediate many adrenaline and noradrenaline (NA) actions and participate in health and disease (Hieble et al., 1995; García-Sáinz et al., 2000). α1B-ARs couple to Gq proteins, which activates phospholipase C, catalyzing inositol trisphosphate and diacylglycerol generation, calcium signaling, and protein kinase C (PKC) activation. α1B-ARs function is tightly regulated and their signaling is markedly attenuated upon sustained agonist activation (homologous desensitization), as well as in response to unrelated agents (heterologous desensitization) acting either through other G protein-coupled receptors, receptor tyrosine kinases, and nuclear receptors, or by direct stimulation of protein kinases such as PKC (García-Sáinz et al., 2000, 2011). Agonist-induced α1B-AR phosphorylation and desensitization is mainly the result of the action of G protein-coupled receptor kinases, whereas second messenger–activated kinases, particularly PKC, are the major mediators of the heterologous process (Diviani et al., 1996, 1997; García-Sáinz et al., 2000, 2011; Castillo-Badillo et al., 2012). Receptor phosphorylation appears to be an early step in desensitization, as it triggers recruitment of β-arrestins, clathrin, and other proteins, such as Rab GTPases, which participate in receptor internalization (Lefkowitz, 2013). α1B-AR internalization seems to involve an initial sequestration away from the plasma membrane followed by endocytosis [reviewed in Toews et al. (2003)].
Rab proteins are monomeric GTPases, considered key regulators of vesicular transport (Zerial and McBride, 2001; Stenmark, 2009). There are more than 60 different types that localize to the cytoplasmic face of specialized membranous organelles (Zerial and McBride, 2001; Stenmark, 2009). These GTPases facilitate docking, fusion, and vesicle transport along the cytoskeleton (Zerial and McBride, 2001; Stenmark, 2009). Specifically, Rab5 and Rab9 are key elements in the crossroads defining whether receptors rapidly recycle back to the plasma membrane or whether they are targeted to late endosomes for slow recycling and/or degradation. Rab5 is an essential regulator of endocytosis; it localizes to endosome and mediates early membrane fusion processes (Zhu et al., 2004). In contrast, Rab9 is present in late endosomes, it is mainly involved in cargo transport between these vesicles and the trans-Golgi network (Choudhury et al., 2005; Schwartz et al., 2007; Kloer et al., 2010).
Rab proteins regulate a number of G protein-coupled receptors (GPCRs), including β1-ARs (Filipeanu et al., 2006; Gardner et al., 2011), β2-ARs (Moore et al., 2004; Filipeanu et al., 2006; Parent et al., 2009; Dong et al., 2010; Lachance et al., 2011; Hammad et al., 2012), α2A-ARs (Li et al., 2012), α2B -ARs (Dong et al., 2010; Li et al., 2012), angiotensin II AT1 receptors (Hunyady et al., 2002; Seachrist et al., 2002; Li et al., 2010; Esseltine et al., 2011; Szakadáti et al., 2015), bradykinin B2 receptors (Charest-Morin et al., 2013), thromboxane A2 receptors (Hamelin et al., 2005), the calcium-sensing receptor (Reyes-Ibarra et al., 2007), and metabotropic glutamate receptors of the 1a subtype (mGluR1a) (Esseltine et al., 2012). In some of these cases, there is evidence indicating direct binding of Rab proteins to specific GPCR domains mainly located at their carboxyl termini (Parent et al., 2009; Dong et al., 2010). It has also been observed that G protein βγ subunits interact with Rab11a and that they colocalize at recycling endosomes in response to cell activation of lysophosphatidic acid receptors (García-Regalado et al., 2008). However, little is known about the mechanism and functional repercussions of interaction between Rab9 and GPCRs.
The possible role of Rab proteins in the function, desensitization, and internalization of α1-ARs has received little attention. It has been reported that Rab1 plays a key role in the transport of α1-ARs from the endoplasmic reticulum to the plasma membrane (anterograde traffic) (Filipeanu et al., 2006). Regarding receptor internalization (retrograde transport), we recently reported that α1B-ARs differentially interact with Rab5 and Rab9 during homologous (agonist-induced) and heterologous desensitizations [induced by activation of S1P1 sphingosine 1-phosphate (S1P) receptors], i.e., noradrenaline triggers a strong and progressive association of these receptors with Rab5, and very little with Rab9, whereas activation of S1P1 receptors essentially induced the opposite effect (Castillo-Badillo et al., 2015).
The aims of the present work were to study the roles of Rab9 and PKC in α1B-AR trafficking and desensitization. To investigate receptor-Rab interaction, we employed confocal microscopy and Förster resonance energy transfer (FRET), using Discosoma spp. red fluorescent protein (DsRed)-tagged α1B-ARs and enhanced green fluorescent protein (EGFP)-tagged Rab proteins, as described previously (Castillo-Badillo et al., 2015). Our results provide new insights into the roles of PKC and Rab9 in modulating α1B-AR vesicular traffic and signaling.
Materials and Methods
Materials.
PMA (phorbol 12-myristate 13-acetate; PubChem CID 27924), dl-propranolol (1-naphthalen-1-yloxy-3-(propan-2-ylamino)propan-2-ol; PubChem CID 4946), NA (noradrenaline, norepinephrine; 4-[(1R)-2-amino-1-hydroxyethyl]benzene-1,2-diol; PubChem CID 439260), S1P (sphingosine 1-phosphate; [(E,2S,3R)-2-amino-3-hydroxyoctadec-4-enyl] dihydrogen phosphate; PubChem CID 5283560), bisindolylmaleimide I (2-(1-(3-dimethylaminopropyl)indol-3-yl)-3-(indol-3-yl)maleimide; PubChem CID 6419775), hispidin (6-[(E)-2-(3,4-dihydroxyphenyl)ethenyl]-4-hydroxypyran-2-one, PubChem CID 54685921), and DNA purification kits were purchased from Sigma-Aldrich (St. Louis, MO). Polyethyleneimine (PubChem CID 6453551) was obtained from Polysciences, Inc. (Warrington, PA), Gö 6976 (12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole; PubChem CID 3501) from Calbiochem/Merck-Millipore (Billerica, MA), and Lipofectamine from Invitrogen/ThermoFisher Scientific (Waltham, MA). Sulfo-NHS-SS-biotin (sodium; 1-[3-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-day]imidazol-4-yl]pentanoylamino]ethyldisulfanyl]propanoyloxy]-2,5-dioxopyrrolidine-3-sulfonate; PubChem CID 71571496) was obtained from Pierce/ThermoFisher Scientific, and the Vectastain kit, from Vector Laboratories (Burlingame, CA). Primary antibodies against the PKC isoforms and β-actin were from Santa Cruz Biotechnology (Dallas, TX) and chemiluminescence kits were from Pierce/ThermoFisher Scientific (Hernández-Méndez et al., 2014; Morquecho-León et al., 2014). Fura-2/AM [bis(acetoxymethyl) 2,2′-((2-(5-((acetoxymethoxy)carbonyl)oxazol-2-yl)-5-(2-(2-(bis(2-(acetoxymethoxy)-2-oxoethyl)amino)-5-methylphenoxy)ethoxy)benzofuran-6-yl)azanediyl)diacetate]; PubChem 3364574), Dulbecco’s modified Eagle’s medium, fetal bovine serum albumin, trypsin, antibiotics, and other reagents employed for cell culture were from Life Technologies/ThermoFisher Scientific. Other reagents were from previously described sources (Castillo-Badillo et al., 2012, 2015).
Plasmids.
The human α1B-AR coding sequence was subcloned into pDsRed-monomer N-1 (Clontech Laboratories Inc., Palo Alto, CA), as described previously (Castillo-Badillo et al., 2015). Proper insertion was confirmed by restriction analysis and sequencing at the Molecular Biology Unit of our Institute. A plasmid coding for α1B-AR DsRed containing S396A and S402A substitutions was synthesized commercially for our group by Mutagenex Inc. (Somerset, NJ). EGFP-tagged Rab5, Rab9 wild-type, and Rab9 dominant-negative GTPases were generated by Dr. Robert Lodge (Institut de Recherches Cliniques de Montréal, Montreal, Canada) (Hunyady et al., 2002) and generously provided to us. All of these constructs are functional, as described previously (Castillo-Badillo et al., 2015).
Cells and Transfection.
Human embryonic kidney 293 (HEK293) cells (American Type Culture Collection, Manassas, VA) were cultured in Dulbecco’s modified Eagle’s medium containing glutamine and high-glucose supplemented with 10% fetal bovine serum, 100 μg/ml streptomycin, 100 IU/ml penicillin, and 0.25 μg/ml amphotericin B, at 37°C, under a 95% air/5% CO2 atmosphere. To study Rab-receptor interaction, plasmids for expression of the EGFP-tagged Rab proteins and DsRed-tagged α1B-ARs were cotransfected employing polyethyleneimine (Hsu and Uludağ, 2012); experiments were carried out 72 hours post-transfection. To assess intracellular calcium concentration, experiments were performed 48 hours post-transfection. DDT1-MF2 cells were obtained from the American Type Culture Collection and were cultured in Dulbecco’s modified Eagle’s medium as described (Molina-Muñoz et al., 2006). Transfection with plasmids for expression of the EGFP-tagged Rab proteins was performed employing Lipofectamine 2000. Because transfection efficiency was low, cells were transfected four times (Yamamoto et al., 1999) with a 1-week interval and subjected to selection in media containing G418 (600 μg/ml). After this time, cells were maintained in buffer containing 300 μg/ml of G418. Using this protocol, the percentage of EGFP-tagged Rab proteins expressing-cells was ∼60–70%, as evidenced by fluorescence.
Confocal Immunofluorescence Microscopy.
This procedure was described in detail previously (Castillo-Badillo et al., 2015). Briefly, cells were cultured in glass-bottomed Petri dishes and treated with the agents and for the times indicated and immediately fixed with 4% paraformaldehyde. When NA was employed, 0.1 μM propranolol was added to block endogenous β-adrenergic receptors. Images of four to six independent experiments using different cell cultures were obtained employing a FluoView Confocal microscope model FV10i [LD laser, 405 nm (18 mW), 473 nm (12.5 mW), 635 nm (10 mW), 559 nm (Olympus, Center Valley, PA)] with a water-immersion objective (60×). EGFP was excited at 480 nm, and the emitted fluorescence was detected at 515–540 nm; the red fluorescent protein DsRed was excited at 557 nm and the fluorescence emitted was detected at 592 nm.
Förster Resonance Energy Transfer.
Interaction between α1B-ARs and Rab proteins was analyzed using FRET by means of the sensitized-emission method, employing a confocal microscope equipped with an automated laser spectral scan FV10i Olympus, as described (Castillo-Badillo et al., 2015). Expression of EGFP- and DsRed-tagged proteins was confirmed for each experiment. EGFP was excited at 480 nm and emitted fluorescence was detected at 515–540 nm; DsRed was excited at 557 nm and emitted fluorescence detected at 592 nm. For the FRET channel analysis, EGFP (but not DsRed) was excited and fluorescence was detected at 592 nm. Images of four to six independent experiments using different cell cultures and at least eight photographs per sample were captured to estimate the interaction of Rab proteins with α1B-ARs by the FRET index after treatment with either agent. The FRET index (that eliminates “bleed-through” and “false” FRET) was quantified using ImageJ software and the “FRET and Colocalization Analyzer” plug-in (Hachet-Haas et al., 2006). This plug-in functions with 8-bit images (Hachet-Haas et al., 2006) and allows supervised computation of the FRET index by means of a “pixel by pixel” method. ImageJ version 1.47b software was obtained from the National Institutes of Health website (Rasband WS. ImageJ, U.S. National Institutes of Health, Bethesda, Maryland, USA, imagej.nih.gov/ij/, 1997–2012.).
Intracellular Calcium Determinations.
Intracellular calcium concentration was assessed as previously described (Hachet-Haas et al., 2006). Cells were serum-starved for 2 hours, then loaded with 2.5 μM of the fluorescent Ca2+ indicator Fura-2/AM in Krebs-Ringer-HEPES containing 0.05% bovine serum albumin, pH 7.4, for 1 hour at 37°C, and subsequently washed three times to eliminate unincorporated dye. Fluorescence measurements were carried out at 340- and 380-nm excitation wavelengths and at a 510-nm emission wavelength, with a chopper interval set at 0.5 seconds, utilizing an Aminco-Bowman Series 2 Luminescence Spectrometer (Rochester, NY). Intracellular calcium levels were calculated according to Grynkiewicz et al. (1985).
Cleavable Biotin Protection Assay.
Experiments were performed as described previously (Reyes-Ibarra et al., 2007). In brief, cells were treated for 30 minutes at 4°C with 30 μg/ml of disulfide-cleavable biotin (sulfo-NHS-SS-biotin), washed to remove free biotin, and prewarmed at 37°C in serum-free culture medium. Incubation was continued for 15 minutes in the absence or presence of the agents tested. After this, cells were washed and subjected to biotin removal from the plasma membrane by incubating the cells in glutathione reducing buffer (50 mM glutathione, 75 mM NaCl, 75 mM NaOH, and 1% fetal bovine serum) for 20 minutes at 4°C. Unreacted glutathione was quenched using iodoacetamide buffer (50 mM iodoacetamide and 1% bovine serum albumin in phosphate-buffered saline); then cells were washed again and lysed. Immunoprecipitation was performed using an antiserum generated in our laboratory against DsRed (Castillo-Badillo et al., 2015). Samples were denatured in Laemmli’s sample buffer (Laemmli, 1970) under nonreducing conditions, subjected to SDS-PAGE, and transferred to nitrocellulose. Biotinylated α1B-ARs were visualized using Vectastain ABC kit (Vector Laboratories) and chemiluminescence.
Western Blot Assays.
Western blotting was performed as described previously (Hernández-Méndez et al., 2014; Morquecho-León et al., 2014). In brief, cells were washed and lysed, lysates were centrifuged and 12,700g, for 15 minutes, and proteins in supernatants were subjected to SDS-PAGE. Proteins were electrotransferred onto nitrocellulose membranes and subjected to immunoblotting. Incubation with primary antibodies was for 12 hours at 4°C and with secondary antibodies for 1 hour at room temperature. Chemiluminescence kits were employed and signals were quantified by densitometric analysis using the Scion Image from Scion Corporation (Frederick, MD).
Statistical Analysis.
When data were normalized, the reference average of each individual experiment (i.e., the 100% value, usually resulting from data from three to four experiments) was used to calculate all the data. Statistical comparison between two paired groups was performed by the Student t test, and when three or more conditions were compared, ANOVA with Bonferroni’s post-test was used. In both cases the software included in the GraphPad Prism program was employed.
Results
PKC Activation Triggered α1B-AR Traffic to Early and Late Endosomes.
To assess the effect of the pharmacological activation of PKC in α1B-AR traffic, we transiently cotransfected HEK293 cells with: 1) a plasmid coding for the DsRed-α1B-ARs and 2) with those coding for either EGFP-Rab5 or EGFP-Rab9. As depicted in Fig. 1A, EGFP-Rab5 and DsRed-α1B-ARs were both present in the cells (see EGFP and DsRed channels). We then measured FRET index intensities upon stimulation with the PKC activator PMA. First, we evaluated receptor interaction with EGFP-Rab5, an early endosome marker. As shown in Fig. 1B, 1 μM PMA induced α1B-AR-Rab5 FRET after 2 minutes and this decreased toward baseline afterward. Similar kinetics were observed with 1 μM S1P, a positive control for α1B-AR heterologous desensitization (Castillo-Badillo et al., 2015). The data indicate that PKC pharmacological activation triggers a receptor traffic route similar to that elicited by receptor-mediated (i.e., through S1P1 receptors) heterologous desensitization. In contrast, 10 μM NA, a positive control for α1B-AR homologous desensitization, rapidly and markedly induced FRET, which continued increasing afterward (Fig. 1B), as previously reported (Castillo-Badillo et al., 2015). Representative images obtained from cells incubated for 15 minutes without any agent (Baseline) or in the presence of 1 μM PMA, 1 μM S1P, or 10 μM NA for 15 minutes are presented in Fig. 1A. Similarly, representative FRET index images corresponding to the time-course of α1B-AR -Rab5 interaction are depicted in Supplemental Fig. S1. As shown, receptors appear to be directed mainly toward early endosomes during homologous desensitization and only transiently interact with this Rab5 during the heterologous processes.
α1B-AR-Rab5 interaction. (A) Images of cells coexpressing DsRed-α1B-ARs and EGFP-Rab5. Cells were incubated for 15 minutes in the absence of any agent (Baseline, upper row) or presence of 1 μM PMA (PMA, second row), 1 μM sphingosine 1-phosphate (S1P, third row) or 10 μM noradrenaline plus 0.1 μM propranolol (NA, fourth row). Cells were fixed and observed in a fluorescence confocal microscope. The following images are presented: EGFP fluorescence (EGFP was excited and its fluorescence recorded; first column), DsRed fluorescence (DsRed was excited and its fluorescence recorded; second column), “FRET channel” (EGFP was excited, the laser to excite DsRed remained off, and DsRed fluorescence was recorded; third column), and “FRET index” (images processed with the “FRET and Colocalization Analyzer,” fourth column). Scale bars: 15 μm. (B) Quantitative analysis of the α1B-adrenergic receptor-Rab5 interaction time-course. Plotted are the means and vertical lines representing the S.E.M of six images for each condition; three experiments were performed utilizing different cell preparations. PMA (1 μM red line and symbols), S1P 1 (1 μM, brown line and symbols), and NA (10 μM plus 0.1 μM propranolol, blue line and symbols). *P < 0.001 versus their respective baseline (Time 0); **P < 0.001 versus their respective baseline (Time 0) and P < 0.001 versus treatments with S1P or PMA for the same time.
Similar experiments were performed to study α1B-AR- Rab9 interaction and the pattern observed was completely different, as depicted in Fig. 2A. Representative images presented clear expression (fluorescent signal) of both α1B-AR and Rab9 (DsRed and EGFP channels, respectively). FRET was detected after a 15-minute stimulation with 1 μM PMA or 1 μM S1P treatments but not in the presence of 10 μM NA (FRET and FRET index channels). The time-course of these effects (Fig. 2B) exhibited significant increase in FRET signal after 2- and 5-minute stimulation with 1 μM S1P, reaching a maximum at 15 minutes [positive control (Castillo-Badillo et al., 2015)]. PMA mimicked this effect and NA, employed as a negative control, induced no significant effect (Fig. 2B). Representative FRET index images corresponding to the time-course of α1B-AR -Rab9 interaction are depicted in Supplemental Fig. S2.
α1B-AR-Rab9 interaction. (A) Images of cells coexpressing DsRed-α1B-ARs and EGFP-Rab9. Other indications as in Fig. 1. (B) Quantitative analysis of the α1B-adrenergic receptor-Rab9 interaction time-course. Plotted are the means and vertical lines representing the S.E.M of five images for each condition for each of the five experiments performed using different cell preparations. *P < 0.001 versus their respective baseline (Time 0) and also P < 0.001 versus treatments with S1P or PMA for the same time. Other indications as in Fig. 1.
Late Endosome α1B-AR Traffic Required PKC Activity and Receptor Phosphorylation.
The data described previously suggested that PKC activation induced α1B-AR trafficking to late endosomes. To further define the role of PKC in these processes, we employed two approaches: 1) the use of a broad-spectrum PKC inhibitor, bisindolylmaleimide I, and 2) PKC downregulation by overnight exposure to PMA (Krug and Tashjian, 1987; Hernández-Méndez et al., 2014; Morquecho-León et al., 2014). In the first approach, HEK293 cells, cotransfected with EGFP-Rab9 and DsRed-α1B-AR, were stimulated with 1 μM PMA, 1 μM S1P, or 10 μM NA for 15 minutes in the absence or presence of 1 μM bisindolylmaleimide I. We then assessed α1B-AR-Rab9 interaction using FRET. As expected, PMA and S1P induced a clear FRET signal in cells incubated in the absence of bisindolylmaleimide I (Fig. 3, A and C). However, the α1B-AR-Rab9 FRET signal was abolished in cells incubated with the inhibitor (Fig. 3, B and C). FRET levels were similar between nonstimulated cells (Baseline) and the negative control (NA), as anticipated (Fig. 3A). In the second approach, cells were incubated overnight with 1 μM PMA to downregulate PKC isoforms (Krug and Tashjian, 1987; Melnikov and Sagi-Eisenberg, 2009; Lum et al., 2013; Hernández-Méndez et al., 2014; Morquecho-León et al., 2014); this treatment markedly reduced the total amount of PKC α, βI (both conventional isoforms), and ε (novel isoform), but not ζ (atypical isoform); no change was observed either in the amount of the loading control, β-actin (Supplemental Fig. S3). The results of both approaches were essentially identical, i.e., the overnight PMA-treatment abolished PMA- and S1P-induced α1B-AR-Rab9 FRET (Fig. 3, C and D), indicating that PKC activity is necessary to direct α1B-AR to late endosomes during heterologous desensitization.
Role of PKC on α1B-AR-Rab9 interaction. (A) Quantitative analysis of the α1B-adrenergic receptor-Rab9 interaction. Cells were preincubated in the absence of any agent (None, open bars), in the presence of 1 μM bisindolylmaleimide I (BIM, hatched bars) for 15 minutes, or the night before the experiment with 1 μM PMA [PMA-ON (overnight), black bars]; after this pretreatment cells were challenged for 15 minutes with vehicle (Baseline), 1 μM PMA, 1 μM S1P, or 10 μM NA (plus propranolol) and the samples were fixed for image acquisition. Plotted are the means and vertical lines representing the S.E.M of five images for each condition for each of the four experiments performed using different cell preparations. *P < 0.001 versus their respective baseline. (B, D) Representative images of cells pretreated with bisindolylmaleimide for 15 minutes [BIM; (B)] or with PMA overnight [PMA-ON (D)]. Other indications as in Fig. 1. (C) Representative FRET index images of cells preincubated in the absence of any agent (None), in the presence of 1 μM bisindolylmaleimide (BIM) for 15 minutes or overnight with 1 μM PMA [(PMA-ON) black bars]; after this pretreatment cells were challenged for 15 minutes with vehicle (Baseline), 1 μM PMA, 1 μM S1P, or 10 μM NA (plus propranolol) and the samples were fixed for image acquisition. Other indications as in Fig. 1.
The previous data prompted us to explore whether PKC phosphorylation sites present in α1B-ARs participate in this process. Elegant work has already identified these sites at the α1B-AR carboxyl tail at S396 and S402 (Diviani et al., 1997). To accomplish this, we transiently cotransfected HEK293 cells with EGFP-Rab9 and wild-type DsRed-α1B-ARs or the PKC phosphorylation site–defective double mutant (S396A, S402A), and receptor-Rab interactions were determined using FRET. It was observed that, using the α1B-AR mutant, PMA and S1P were able to elicit a clear FRET response, but it was consistently smaller than that detected using the wild-type receptor (Fig. 4, A–C). These data suggest that phosphorylation in S396 and/ or S402 participate in α1B-AR-Rab9 association and traffic.
Role of α1B-AR PKC phosphorylation sites on α1B-AR-Rab9 interaction. (A) Quantitative analysis of the α1B-adrenergic receptor-Rab9 interaction. Cells expressing wild-type DsRed-α1B-ARs (open bars, WT) or the PKC phosphorylation site-defective DsRed-α1B-AR mutants (S396A, S402A) (hatched bar, Mut) were challenged for 15 minutes with vehicle (Baseline), 1 μM PMA, 1 μM S1P, or 10 μM NA (plus propranolol). Plotted are the means and vertical lines representing the S.E.M of six images for each condition for each of the four experiments performed using different cell preparations. *P < 0.001 versus their respective baseline; **P < 0.05 versus its respective baseline and P < 0.05 versus PMA wild-type; ***P < 0.001 versus its respective baseline and P < 0.05 versus S1P wild-type. (B) Representative images of cells expressing the PKC phosphorylation sites-defective DsRed-α1B-AR mutant (S396A, S402A) challenged for 15 minutes with vehicle (Baseline), 1 μM PMA, 1 μM S1P, or 10 μM NA (plus propranolol). Other indications as in Fig. 1. (C) Representative FRET index images of cells expressing DsRed-α1B-ARs wild-type (WT) or the PKC phosphorylation sites-defective mutant (S396A, S402A) (Mut) challenged for 15 minutes with vehicle (Baseline), 1 μM PMA, 1 μM S1P, or 10 μM NA (plus propranolol). Other indications as in Fig. 1.
Rab9 -α1B-AR Interaction Was GTPase Activity-Dependent.
To define whether Rab9 GDP/GTP exchange was necessary for its association with α1B-ARs and their subsequent transport to late endosomes, we evaluated changes in FRET signal in the presence of a GDP-locked dominant-negative form of Rab9 (Hunyady et al., 2002). It was observed that overexpression of this mutant allowed no increase in FRET signal in response to PMA or S1P stimulation, compared with positive controls expressing wild-type Rab9 (Fig. 5, A–C). Data indicate that Rab9 associates with α1B-ARs in a process dependent on its GTPase activity. As anticipated, no FRET signal was detected upon NA stimulation (Fig. 5, A–C), emphasizing that this process mainly affects vesicular traffic elicited during heterologous α1B-AR desensitization.
Role of Rab9 GTPase activity on α1B-AR-Rab9 interaction. (A) Quantitative analysis of the α1B-AR-Rab9 interaction. Cells expressing wild-type DsRed-α1B-ARs and either EGFP-Rab9 wild-type (open bars, Rab9-WT) or the GDP-locked dominant-negative mutant (hatched bar, Rab9-GDP) were challenged for 15 minutes with vehicle (Baseline), 1 μM PMA, 1 μM S1P, or 10 μM NA (plus propranolol). Plotted are the means and vertical lines representing the S.E.M of six images for each condition for each of the four experiments performed using different cell preparations. *P < 0.001 versus their respective baseline. (B) Representative images of cells expressing the GDP-locked Rab9 mutant (Rab9-GDP) were challenged for 15 minutes with vehicle (Baseline), 1 μM PMA, 1 μM S1P, or 10 μM NA (plus propranolol). Other indications as in Fig. 1. (C) Representative FRET index images of cells expressing Rab9-WT and the GDP-locked mutant (Rab9-GDP) challenged for 15 minutes with vehicle (Baseline), 1 μM PMA, 1 μM S1P, or 10 μM NA (plus propranolol).
α1B-AR Internalization Was PKC- and Rab9-Modulated.
The possible role of the α1B-AR-Rab9 interaction on receptor internalization was studied by employing confocal microscopy. In cells cotransfected with DsRed-α1B-AR and wild-type EGFP-Rab9, the receptor was mainly localized at the plasma membrane with some fluorescence also observed in intracellular vesicles (Baseline) (Fig. 6, upper row; representative images evidencing expression of EGFP-Rab proteins are presented in Supplemental Fig. S4). This localization drastically changed (i.e., membrane delineation was markedly decreased and fluorescence accumulation into intracellular vesicles was observed) upon stimulation for 15 minutes with PMA, S1P, or NA (Fig. 6, first column). Fluorescence accumulation in intracellular vesicles was a clearer index of internalization and its quantitation is presented in Fig. 7A. It can be observed that PMA and NA increased intracellular fluorescence ∼2-fold and S1P slightly less. The time-course of these actions is shown in Supplemental Fig. S5; PMA and NA rapidly increased receptor internalization, whereas the action of S1P took place more slowly. Internalization was confirmed using the disulfide-cleavable biotin protection assay. As shown in Supplemental Fig. S6, the amount of biotin-labeled α1B-ARs located intracellularly increased in cells incubated with PMA, S1P, or NA for 15 minutes.
Internalization of α1B-ARs. Representative images (red channel) of cells expressing DsRed α1B-ARs (first four columns) and the PKC phosphorylation site-defective DsRed-α1B-AR mutant (S396A, S402A) (fifth column); cells also expressed wild-type EGFP-Rab9 (columns 1–3 and 5) or the GDP-locked mutant (Rab9-GDP, fourth column). Preincubation was with 1 μM bisindolylmaleimide I for 15 minutes (BIM, second column) or overnight with 1 μM PMA (PMA-ON, third column). Cells were challenged for 15 minutes with vehicle (Baseline), 1 μM PMA, 1 μM S1P, or 10 μM NA (plus propranolol). Images showing Rab9 expression are presented in Supplemental Fig. S4).
Quantitation of intracellular fluorescence as an index of α1B-AR internalization. Data obtained from cells expressing DsRed α1B-ARs (A–D) or the PKC phosphorylation site-defective DsRed-α1B-AR mutant (S396A, S402A) (E); cells also expressed wild-type EGFP-Rab9 (A–C and E) or the GDP-locked mutant [Rab9-GDP (D)]. Preincubation was with 1 μM bisindolylmaleimide I for 15 minutes [BIM (B)] or overnight with 1 μM PMA [PMA-ON (C)]. Cells were challenged for 15 minutes with vehicle (B, Baseline), 1 μM PMA, 1 μM S1P, or 10 μM NA. In all panels, the means are plotted with vertical lines representing the S.E.M. of five to six experiments using different cell preparations; in each experiment five different images were analyzed (i.e., 25–30 samples in each condition). *P < 0.001 versus B (baseline); **P < 0.01 versus B (Baseline).
Employing fluorescence detection, it was observed that when cells were incubated with bisindolylmaleimide I, receptor fluorescence remained at the plasma membrane after treatments with PMA or S1P. In contrast, internalization was observed in NA-treated cells (Fig. 6, second column; Fig. 7B). Essentially the same results were obtained both in cells in which PKC was downregulated by overnight PMA treatment (Fig. 6, third column; Fig. 7C) as well as when the GDP-locked dominant-negative form of Rab9 was expressed (Fig. 6, fourth column; Fig. 7D), i.e., PMA- and S1P-induced internalizations were markedly attenuated, whereas that triggered by NA was not affected. These data unraveled PKC and Rab9 GTPase activities relevant for α1B-AR internalization during heterologous desensitization. The role of the PKC phosphorylation sites identified was studied employing the DsRed-α1B-AR (S396A, S402A) mutant cotransfected with wild-type EGFP-Rab9. Interestingly, this mutant receptor internalized in response to PMA, S1P, and NA.
Rab9 Regulates α1B-AR Desensitization.
To determine the repercussions of α1B-AR-Rab9 interaction in calcium signaling, cells were transfected with plasmids coding for DsRed-α1B-AR alone or together with plasmids coding for EGFP-Rab proteins. NA-triggered intracellular calcium increases were observed in all the conditions studied. As anticipated, in cells expressing α1B-ARs and only the endogenous Rab proteins, PMA induced a marked desensitization of the response to NA (∼70–80%) (Fig. 8A), whereas transfection of the different Rab proteins decreased the magnitude of such desensitization (Fig. 8, B–D). In particular, expression of the dominant negative mutant, Rab9-GDP, resulted in marked resistance to the action of PMA (i.e., the action of the active phorbol ester was not statistically significant) (Fig. 8C). In contrast, expression of the Rab5-GDP dominant-negative mutant did not alter the effect of PMA to such an extent (Fig. 8D). NA-induced calcium response was only marginally affected by PMA in cells expressing the α1B-AR (S396A, S402A) mutant (Fig. 8E).
Effect of Rab9 expression on NA-induced α1B-AR-mediated intracellular calcium increases and PMA action. Cells expressing either DsRed-α1B-ARs and 1) no exogenous Rab protein [No Rab, vector (A)]; 2) wild-type EGFP-Rab9 [Rab9-WT (B)]; 3) the GDP-locked EGFP-Rab9 mutant [Rab9-GDP (C)]; 4) the GDP-locked EGFP-Rab5 mutant [Rab5-GDP (D)]; or the DsRed-α1B-AR mutant (S396A, S402A) with no exogenous Rab protein (E) were used. Cells were preincubated for 5 minutes in the absence of any agent (open bars) or the presence of 1 μM PMA (shaded bars); cells were then challenged with 10 μM NA (plus propranolol). Plotted are the means and vertical lines representing the S.E.M of eight to ten determinations using different cell preparations. *P < 0.001 versus absence of PMA, **P < 0.01 versus absence of PMA.
Studies Using DDT1-MF2 Cells.
We explored the possibility that some of the actions observed using transfected HEK293 cells could also be detected in a cell line that endogenously expressed α1B-ARs. DDT1-MF2 cells abundantly express these receptors and have been used as a cellular model to study their actions and regulation (Leeb-Lundberg et al., 1985; García-Sáinz et al., 2004). In addition, the ability of active phorbol esters to desensitize α1B-ARs was first shown in isolated rat hepatocytes (Corvera and García-Sáinz, 1984; Corvera et al., 1986), but their actual receptor phosphorylation was initially shown using DDT1-MF2 cells (Leeb-Lundberg et al., 1985). We confirmed the ability of PMA to block α1B-AR action and studied the ability of a series of PKC inhibitors. PMA-induced α1B-AR desensitization was essentially blocked by bisindolylmaleimide I (general PKC inhibitor) and Gö 6976 [PKC α- and β-selective inhibitor (Martiny-Baron et al., 1993)] and was reduced by hispidin [PKC β-selective inhibitor (Gonindard et al., 1997)] (Supplemental Fig. S7A); the inhibitors themselves did not alter NA action (data not shown). As expected, overnight treatment with PMA essentially abolished the ability of acute addition of the phorbol ester to desensitize NA action in DDT1-MF2 cells (Supplemental Fig. S7B). We also tested the effect of expression of the Rab-EGFP constructs on the ability of PMA to desensitize α1B-ARs in DDT1-MF2 cells. Transfection of these constructs was not very efficient in these cells; therefore, we had to use multiple transfections and the selection protocol described under Material and Methods, to obtain cells with 60–70% Rab-EGFP expression, as evidenced by fluorescence microscopy. Using these cells, we observed that expression of Rab9 (Fig. 9B) and in particular the Rab9-GDP mutant (Fig. 9C) decreased PMA-induced α1B-AR desensitization; expression of Rab5-GDP did not affect this PMA action (Fig. 9D); which is remarkably similar to what was observed using HEK293 cells (Fig. 8).
Effect of Rab9 expression on NA-induced α1B-AR-mediated intracellular calcium increases and PMA action. DDT1-MF2 cells transfected to express: 1) no exogenous Rab protein [No Rab (A)]; 2) wild-type EGFP-Rab9 [Rab9-WT (B)]; 3) the GDP-locked EGFP-Rab9 mutant [Rab9-GDP (C)]; and 4) the GDP-locked EGFP-Rab5 mutant [Rab5-GDP (D)] were used. Cells were preincubated for 5 minutes in the absence of any agent (open bars) or the presence of 1 μM PMA (shaded bars); cells were then challenged with 10 μM NA. Plotted are the means and vertical lines representing the S.E.M of six to nine determinations using different cell preparations. *P < 0.001 versus absence of PMA.
Discussion
The possible role(s) of Rab GTPases in α1-AR action, vesicular traffic, and its regulation has received little attention. To the best of our knowledge, our previous publication (Castillo-Badillo et al., 2015) is the only one dealing with retrograde traffic, thus far. Data in our present work indicate that direct activation of PKC with phorbol esters induces Rab9-α1B-AR interaction with great similarity to that observed with S1P (Castillo-Badillo et al., 2015). In other words, similar patterns of α1B-AR-Rab interaction take place when PKC is activated either physiologically or by pharmacological means. In contrast, this was not observed when cells were stimulated with the natural agonist NA, emphasizing differences between homologous and heterologous desensitizations. Rab9-α1B-AR interaction and internalization are blocked by bisindolylmaleimide I, a PKC inhibitor, and by the downregulation of this enzyme (induced by overnight incubation with PMA). In both cases, Rab9-α1B-AR interaction required Rab9 GTPase activity. PMA and S1P induced α1B-AR internalization that was markedly diminished by the same treatments, i.e., PKC inhibition, PKC downregulation, and the expression of a GDP-locked dominant-negative Rab9 mutant. These data are consistent with the current idea that PKC is a major mediator of α1B-AR heterologous desensitization, including receptor phosphorylation and internalization; this can be induced by S1P (Castillo-Badillo et al., 2012, 2015) and by a large variety of hormones and neurotransmitters acting through different families of receptors [see Castillo-Badillo et al. (2012, 2015); García-Sáinz et al. (2000, 2011); and references therein]. A working model is presented in Fig. 10. The PKC downregulation studies and the pharmacological profile of the PKC inhibitors used suggest that conventional PKC isoforms are participants in the actions described, which is consistent with what we have observed for LPA1 and S1P1 receptors (Hernández-Méndez et al., 2014; Morquecho-León et al., 2014).
Working model for the possible roles of PKC and Rab proteins in receptor internalization.
Major issues include how the cellular machinery “senses” when a receptor is internalized in response to agonist activation or to other processes and how this event is linked to the different internalization pathways. Although this is currently unknown, accumulating evidence suggests that the actual sites phosphorylated in a given receptor can vary (Torrecilla et al., 2007; Tobin et al., 2008; Butcher et al., 2014). Such variation has been denominated the “phosphorylation bar code” (Torrecilla et al., 2007; Tobin et al., 2008; Butcher et al., 2014), and it has been proposed that such variations can favor some cellular processes over others, including apoptosis, proliferation, differentiation, or metabolic changes, to mention a few. As indicated, different phosphorylation sites have been detected for PKC and G protein-coupled receptor kinases in α1B-ARs (Diviani et al., 1997); these authors performed their work with the hamster ortholog (sites S394 and S400), which are conserved in human α1B-ARs (corresponding to S396 and S402). When the phosphorylation sites–defective α1B-AR mutant (S396A, S402A) was used, the interaction with Rab9 was clearly diminished but not abolished. These data suggest that such sites participate in the receptor-Rab interaction but may not be the only ones. Internalization of the α1B-AR mutant (S396A, S402A) during heterologous desensitization (i.e., under the action of PMA or S1P) was observed. Interestingly, the ability of PMA to block α1B-AR-mediated increase in intracellular calcium was markedly attenuated. This raises the possibility that other sites or elements could participate in receptor-Rab protein interaction and internalization. It should be kept in mind that differences might exist among receptor orthologs and in cellular models in which the experiments are performed (Tobin et al., 2008; Butcher et al., 2011). Current evidence indicates that Rab5 is involved in the initial endocytosis of vesicles formed from the plasma membrane and their subsequent interaction with early endosomes (Novick and Zerial, 1997; Nielsen et al., 1999; Zerial and McBride, 2001; Bhattacharya et al., 2004); this suggests that agonist-activation targets α1B-ARs to early endosomes and fast recycling back to the plasma membrane. In contrast, Rab9 is a protein characteristic of late endosomes (Novick and Zerial, 1997; Nielsen et al., 1999; Zerial and McBride, 2001; Bhattacharya et al., 2004). This suggests that PKC activation leads initially to a brief interaction of α1B-ARs with early endosomes and subsequent migration to late endosomes. Such differential traffic might have functional consequences.
As already mentioned, Rab proteins appear to regulate the signaling and traffic of a variety of GPCRs and in some cases a direct Rab association with these receptors has been documented, particularly to the GPCR carboxyl termini (Parent et al., 2009; Dong et al., 2010). Esseltine et al. (2011) previously reported that several Rab proteins bind to a common site in the angiotensin II AT1 receptor carboxyl tail and that Rab4, in particular, regulates receptor phosphorylation, desensitization, and resensitization. Regarding the present work, the evidence provided by the FRET analysis indicates that the α1B-AR and Rab9 constructs are in close proximity (i.e., 1–10 nm, the limit for FRET to take place). FRET is a collision-free but distance-dependent photophysical process and one of the few noninvasive approaches currently used to analyze interaction among molecular species and its use in the biologic sciences is expanding exponentially (Shrestha et al., 2015). The FRET-based approach employed in this work was developed for single-cell analysis using confocal fluorescent microscopy and it was also validated using cell suspensions using fluorescence emission scanning (Castillo-Badillo et al., 2015). Other approaches have been successfully used to study GPCR-Rab interactions, including coimmunoprecipitation and colocalization. Using those procedures, we observed that under baseline conditions there was a very strong signal, which did not allow us to detect any changes with the different treatments (Castillo-Badillo et al., 2015). This was confirmed in the present study also and even using cells that endogenously express α1B-ARs (data not shown). Our interpretation is that signaling complexes (signalosomes) are already preformed (detected by coimmunoprecipitation) and that FRET allowed us to detect organization changes within those complexes. More structural work will be necessary to define whether there is a physical interaction between α1B-ARs and Rabs, and the domain(s) and specific residues involved.
Although it is clear that Rab9 modulates α1B-AR traffic to late endosomes, as evidenced by the receptor internalization data, we possess no clear explanation of the fact that Rab9 expression diminished the ability of PMA to desensitize NA-induced α1B-AR-mediated calcium signaling, and why such a diminution was even more pronounced in cells expressing the Rab9 dominant-negative mutant. One possibility is that Rab activity could be involved in PKC-mediated α1B-AR desensitization. However, this does not seem probable because PMA action is usually rather fast, occurring in the range of a few seconds to 2–3 minutes, and precedes any significant receptor internalization. It is more plausible that the interaction of Rab9 with the receptor might represent a physical obstacle for PKC access to the receptor. Were this the case, the difference observed with the dominant-negative mutant could result from a higher level of expression, changes in cell localization, higher affinity for the receptor, or a combination of these or other factors. This aspect remains a puzzling but interesting observation meriting further exploration.
The interaction between GPCRs and Rabs appears to be rather complex, owing to the following: 1) various Rab proteins may compete for the binding sites present in GPCRs (Esseltine et al., 2011), 2) GPCRs are not only “cargo” proteins but also may function as GEFs (guanine nucleotide exchange factors, which facilitate the exchange of GDP for GTP) modifying the activity of Rab proteins (Seachrist et al., 2002), and 3) ligands (agonist/antagonists) might modulate these actions by altering receptor conformation, thus changing GPCR-Rab interactions qualitatively and/or quantitatively (Seachrist et al., 2002). Additionally, receptor activation triggers signaling cascades leading to Rab phosphorylation by protein kinases (Karniguian et al., 1993; Chiariello et al., 1999; Fitzgerald and Reed, 1999; Pavarotti et al., 2012) or prenylation by geranylgeranyltransferases (Lachance et al., 2011); such covalent modifications might alter Rab localization and function. There is already evidence for the PKC-catalyzed phosphorylation of some Rab proteins (Fitzgerald and Reed, 1999; Pavarotti et al., 2012). Preliminary evidence indicates that Rab9 is phosphorylated under baseline conditions and that such phosphorylation was not increased in cells treated with PMA (unpublished observation). Further work will be required to properly define whether Rab9 phosphorylation plays a role and the protein kinases involved.
In summary, our data indicate that PKC pharmacological activation triggered a weak and transient α1B-AR-Rab5 interaction and a progressive and sustained receptor association with Rab9 (suggesting GPCR targeting to late endosomes). The following elements were necessary to elicit such α1B-AR-Rab9 interaction: 1) PKC activity, 2) α1B-AR PKC-mediated phosphorylation at S396 and/or S402 (evidenced by the use of an α1B-AR mutant), and 3) Rab9 GTPase activity. The lack of any of these elements impaired receptor internalization. Finally, we found that the expression of wild-type Rab9 or the dominant-negative mutant of this GTPase altered PMA-induced α1B-AR desensitization.
Acknowledgments
The authors thank: Dr. Rocío Alcántara-Hernández, Juan Barbosa, Aurey Galván, and Manuel Ortínez and to the Computer, Molecular Biology, and Microscope Service Units of our Institute for help and advice. They also thank Maggie Brunner, MA, for style corrections and Dr. Marina Macías-Silva for the donation of some of the reagents used in this work. JAG-S dedicates this work to his advisor, Dr. Victoria Chagoya, on the 50th anniversary of her first international publication.
Authorship Contributions
Participated in research design: Alfonzo-Méndez, Reyes-Cruz, García-Sáinz.
Conducted experiments: Alfonzo-Méndez, Hernández-Espinosa, Carmona-Rosas, Romero-Ávila.
Contributed new reagents or analytic tools: Reyes-Cruz.
Performed data analysis: Alfonzo-Méndez, Hernández-Espinosa, Carmona-Rosas, Romero-Ávila.
Wrote or contributed to the writing of the manuscript: Alfonzo-Méndez, Reyes-Cruz, García-Sáinz.
Footnotes
- Received August 19, 2016.
- Accepted January 9, 2017.
This research was partially supported by grants from Dirección General de Personal Académico-Universidad Nacional Autónoma de México [IN200915] and Consejo Nacional de Ciencia y Tecnología [253156 and Fronteras de la Ciencia 882]. M. A. Alfonzo-Méndez, and D. A. Hernández-Espinosa are students of the Programa de Maestría y Doctorado en Ciencias Bioquímicas, and G. Carmona-Rosas is a student of the Programa de Doctorado en Ciencias Biomédicas-Universidad Nacional Autónoma de México; they are the recipients of fellowships from Consejo Nacional de Ciencia y Tecnología.
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This article has supplemental material available at molpharm.aspetjournals.org.
Abbreviations
- α1B-ARs
- α1B-adrenergic receptors
- DsRed
- Discosoma spp. red fluorescent protein
- EGFP
- enhanced green fluorescent protein
- FRET
- Förster resonance energy transfer
- GPCR
- G protein-coupled receptor
- HEK
- human embryonic kidney
- NA
- noradrenaline
- PMA
- phorbol 12-myristate 13-acetate
- PKC
- protein kinase C
- PMA
- phorbol 12-myristate 13-acetate
- S1P
- sphingosine 1-phosphate
- Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics
References
In this issue
Protein Kinase C Promotes α1B-Adrenoceptor Rab9 Interaction
