Biochemical and Biophysical Research Communications 289, 325–328 (2001) doi:10.1006/bbrc.2001.5981, available online at http://www.idealibrary.com on
Polarized Expression of the GFP-Tagged Rat V 1a Vasopressin Receptor Danae M. Campos,* Carlos E. Reyes,* Jose Sarmiento,* Javier Navarro,† and Carlos B. Gonza´lez* ,1 *Department of Physiology, Universidad Austral de Chile, Valdivia, Chile; and †Membrane Protein Laboratory, Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77555
Received October 22, 2001
We investigated the targeting of the V 1a receptor fused with the green fluorescence protein (V 1aR–GFP) in polarized MDCK cells. Cells expressing V 1aR–GFP displayed binding to vasopressin (AVP) and AVPinduced calcium responses, similar to cells expressing the wild-type V1a receptor. Interestingly, as with the wild-type V 1aR, V 1aR–GFP is preferentially distributed in the basolateral side of MDCK cells as monitored by confocal microscopy. Furthermore, AVP induced internalization of GFP-tagged receptors. Therefore, the GFP-tagged V 1a receptor retains all the sorting signals of the wild-type receptor and offers an excellent system to elucidate the mechanisms of cell trafficking of V 1a receptors. © 2001 Elsevier Science Key Words: vasopressin; V 1a receptor; polarized expression; signaling mechanism.
Trafficking and targeting of newly synthesized proteins are essential for establishing the polarity in secretory epithelial cells. In contradistinction to singlepass transmembrane proteins the sorting of G proteincoupled receptors is not well understood. AVP receptors belong to the superfamily of G protein-coupled receptors. At least three receptor subtypes have been identified, V 1a, V 1b, and V 2, on the basis of their pharmacological profile and signaling mechanism (1). Furthermore, these receptors subtypes are expressed in a tissue-specific fashion. The V 2 receptor mediates the activation of adenylyl cyclase via the Gs family of G proteins, whereas V 1a and V 1b mediate the activation of phospholipase C via Gq family of G proteins (2). The sorting and targeting of AVP receptors is not well defined yet. To determine whether AVP receptors are asymmetrically distributed in the cell we expressed GFP-tagged V 1a receptor in the polarized 1 To whom correspondence and reprint requests should be addressed at Departamento de Fisiologı´a, Universidad Austral de Chile, Casilla 567, Isla Teja, Valdivia 2-5119300, Chile. E-mail:
[email protected].
MDCK cell system, which contain well defined apical and basolateral surfaces. We found that the GFPtagged V 1a receptor is targeted preferentially to the basolateral side of the plasma membrane. MATERIALS AND METHODS Vector constructions. Wild-type V 1a receptor cDNA was amplified by RT-PCR from rat liver mRNA, and subcloned into the expression vector pcDNA3 (Invitrogen) using EcoRI and XbaI sites. The V 1a cDNA was also subcloned into the pEGFP-N1 vector (Clontech) in frame to GFP cDNA. The sequence fidelity of the V 1a–GFP construct was verified by DNA sequencing. To clone the V 1a the following primers were used, the sense was 5⬘-ggaattccaggctctgtacggacagcat-3⬘ and the antisense was 5⬘-gctctagactgcgtgaacgtggggctcaa-3⬘. To subclone the V 1a in frame with the EGFP cDNA we employed the antisense primer, 5⬘-cgggatccgtggagacacaggaatgaatct-3⬘, in order to remove the stop codon of the V 1a. Expression of GFP- tagged V 1a receptor in MDCK cells. MDCK cells were transfected with the wild-type V 1a or the V 1a–GFP constructs using Fugene 6 (Roche). Cells were selected in the presence of 700 g/ml of G418. After 2 weeks, the colonies were isolated, cells were amplified and tested for binding to radiolabeled AVP. Expression of V 1a-GFP was monitored by fluorescence microscopy. Receptor binding assays. For saturation binding assays MDCK cells grown to confluency in 35-mm dishes were incubated with increasing concentrations of [ 3H]arginine AVP (New England Nuclear). For displacement binding assays MDCK cells were incubated with 6 nM tritated AVP in the presence of increasing concentrations of the nonlabeled peptides: [Deamino1, D-Arg8]AVP (dDAVP or desmopressin) (Bachem) and the V1 antagonist d(CH2)5[Tyr2(Me)Tyr9(NH2)]AVP (kindly provided by Professor M. Manning). Data were analyzed by the program Ligand (3). Binding is reported as average of three independent experiments. Statistical analysis was carried out by the Student’s test. Calcium mobilization assays. MDCK cells were resuspended at a concentration of 10 7 cells/ml in a 20 mM Hepes, pH 7.4, buffer containing 140 mM NaCl, 4 mM KCl, 1 mM MgCl 2, 1 mM CaCl 2, 1 mM NaHPO 3, 5 mM glucose, 1 mM probenecid, and 1 mg/ml BSA. The cells were loaded with 5 M Indo-1AM for 30 min at 22°C. Agonist induced-intracellular calcium increase is monitored by measurement of fluorescence using a LS50B Perkin–Elmer spectrofluorometer at a excitation wavelength of 330 nm and emission wavelength of 405 nm. Confocal microscopy. MDCK cells grown to confluency on Anopore permeable membrane (Nunc Inc.) were fixed in 4% paraformaldehyde in PBS for 20 min at 22°C, washed with PBS and mounted for
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0006-291X/01 $35.00 © 2001 Elsevier Science All rights reserved.
Vol. 289, No. 2, 2001
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1
Pharmacological Properties of the Endogenous V 2, Wild-Type V 1a, and GFP-Tagged V 1a Receptors
MDCK MDCK V1 MDCK V1-GFP
Kd (nM)
B max (pmol/10 6 cells)
2.3 ⫾ 0.06 3.8 ⫾ 0.03 1.2 ⫾ 0.05
99 120 180
dDAVP 2.8 ⫾ 0.11
K i (nM) AT1 0.09 ⫾ 0.36 1.40 ⫾ 0.34
fluorescence microscopy (Dako). Preparations were analyzed with a LSM 5 Zeiss laser confocal microscope at an excitation wavelength of 488 nm and an emission of 505 nm.
RESULTS AND DISCUSSION
sitized (Figs. 2A and 2B). The Ca 2⫹ responses mediated by both the wild-type and the GFP-tagged receptors were blocked by the V 1a antagonist (data not shown). Dose–response experiments with cells expressing the GFP-tagged receptor showed an EC 50 of 60 nM. This EC 50 was in agreement with those reported for the wild-type receptor expressed in several cell types (4 – 6). These results indicate that the GFP-tagged receptor display similar functional properties as the wild-type receptor. GFP-Tagged V1a Receptors Are Targeted to the Basal Membrane of MDCK Cells The surface distribution of GFP-tagged receptors in MDCK cells was monitored by laser-scanning microscopy. The xy-scan showed that the fluorescence is
MDCK Cells Expressing GFP-Tagged V 1a Receptors Bind AVP with High Affinity MDCK cells express endogenously V 2 receptors, as demonstrated upon binding to AVP (K d 2.3 ⫾ 0.06 nM and B max 9.9 ⫻ 10 ⫺14 mol per 10 6 cells, Table 1), and selective binding to desmopressin, a specific V 2 receptor agonist. Interestingly, MDCK cells transfected with either cDNAs encoding the wild-type V 1a or the GFPtagged V 1a receptors (Fig. 1B) do not display specific AVP binding mediated by the endogenous V 2 receptor. However, cells transfected with the vector alone exhibited AVP binding similar to untransfected cells. This observation suggests that the AVP binding mediated by the endogenous V 2 receptor is regulated by the expression of the V 1a receptor. Scatchard analysis showed that MDCK cells transfected with the cDNA encoding the V 1a receptor bound AVP with a K d of 3.8 ⫾ 0.03 nM and a B max of 1.2 ⫻ 10 ⫺13 mol per 10 6 cells (Table 1). Competitive displacement of bound AVP with the V 1 antagonist showed a K i of 0.09 ⫾ 0.36 nM (Table 1). Similarly, MDCK cells transfected with cDNA encoding GFP-tagged V 1a receptors bound AVP with a K d of 1.2 ⫾ 0.05 nM and a B max of 1.8 ⫻ 10 ⫺13 mol per 10 6 cells (Table 1, Fig. 1A). Competitive binding with the V 1 receptor antagonist showed a K i of 1.4 ⫾ 0.34 nM (Table 1, Fig. 1B). These findings demonstrate that the GFP-tagged V1a receptor retains the wild type binding pocket for agonists and antagonists. The GFP-Tagged V 1a Receptor Is Functional Cells expressing the wild-type V 1a receptor or the GFP-tagged receptor displayed AVP-induced [Ca 2⫹] responses as monitored by fluorescence spectroscopy with the calcium sensitive dye Indo-1 AM (Figs. 2A and 2B). Cells expressing either receptor exhibited a weak calcium response upon addition to a second dose of agonist, suggesting that both receptors became desen-
FIG. 1. Saturation (A) and competition (B) binding assays in intact MDCK cells expressing the GFP-tagged V 1a vasopressin receptor. Scatchard analysis of the data from A is shown in the inset.
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in the kidney (9). Although, application of AVP to both the apical or basolateral sides of the kidney tubular cells elicits actions mediated by V 1a receptors (10 –14), it is possible that a small proportion of V 1a receptor is present at the apical side in renal tubular cells or that in some tubular cells V 1a receptors are evenly distributed. This view is supported by our studies indicating that while V 1a receptors are located at the basolateral of the collecting duct cells, in connecting cells the receptors are distributed at both basolateral and apical domains (9). AVP-Induced Internalization of the GFP-Tagged Receptor As with the wild-type V 1a receptor the GFP-tagged receptor is internalized upon exposure to AVP as monitored by laser confocal microscopy. After 5–15 min of agonist exposure the GFP-tagged receptor is localized in the subplasmalema compartment, but after 15 min of agonist exposure the GFP-tagged receptor exhibited a perinuclear distribution. After long exposures to the agonist (30 – 60 min) the plasma membrane is mostly depleted of the receptor (Fig. 4). In summary fusion of GFP to the carboxyterminal domain of the V 1a receptor did not alter the binding and functional properties of the receptor. Therefore, this GFP-tagged receptor is suitable system to elucidate the
FIG. 2. Vasopressin-induced calcium responses in MDCK cells expressing the GFP-tagged V 1a receptor (A) the wild-type V 1a receptor (B) and nontransfected cells (C), addition of AVP (1 M) are indicated by arrows. Dose–response curve of vasopressin-induced calcium mobilization in MDCK cells expressing GFP-tagged receptors (D).
mainly restricted to the plasma membrane with only a minor proportion of receptors inside the cell. The z-scan revealed that the fluorescence is confined to the basolateral membrane, specifically to lateral side (Fig. 3). Negligible fluorescence was detected at the apical membrane of MDCK cells. This unique distribution of the GFP-tagged receptor in MDCK is analogous to the distribution of other tagged receptors, including V 2 (7) or the ␣ 2A adrenergic receptors (8), which are targeted to the basolateral domain when they are expressed in MDCK cells. This distribution of the GFP-tagged V 1a receptor is in agreement with the location of the V 1a receptor in the basolateral side of collecting duct cells
FIG. 3. Localization of the GFP-tagged V 1a receptor in MDCK cells transfected with the cDNA encoding for the GPF-tagged V 1a vasopressin receptor using confocal laser microscope. The xy-scan shows the GFP label at the surface of the cell. The z-scans, upper and right pictures show that the GFP-tagged receptor is at the basolateral domain.
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FIG. 4. AVP-induced internalization of the GFP-tagged receptor. MDCK cells transfected with cDNA encoding for the GFP-tagged V 1a receptor were exposed to AVP for 15 min (B) and 60 min (C). There is an increase of the label inside the cells relative to the control (A) in cells treated with AVP (B and C).
mechanisms of trafficking and targeting of V 1a receptors in polarized cells. 7.
ACKNOWLEDGMENTS We thank the expert technical assistant of E. Oyarzun. This work is supported by Grant 8980002 from Fondecyt and DIUAH. The laser confocal microscope was purchased with a grant to the CECS from the Andes Foundation.
8.
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