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CELLULAR AND MOLECULAR

Blockade of G Protein-Coupled Receptors and the Dopamine Transporter by a Transmembrane Domain Peptide: Novel Strategy for Functional Inhibition of Membrane Proteins in Vivo

Departments of Pharmacology (S.R.G., G.V., S.P.L., P.S., B.F.O.), Medicine (S.R.G.), Psychiatry (G.Y.N, P.S.), and Biochemistry (C.W., C.M.D.), University of Toronto, Toronto, Ontario, Canada; and the Centre for Addiction and Mental Health (S.R.G., T.F., B.F.O.), Toronto, Ontario, Canada

Received for publication May 2, 2003
Accepted July 31, 2003.

	Abstract

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G protein-coupled receptors have a core consisting of seven transmembrane -helices that is important in maintaining the structure of the receptor. We postulated that disruption of the transmembrane core may interfere with receptor function. In this study, the function of integral membrane proteins was disrupted in vivo using peptides mimicking their transmembrane domains. A peptide derived from transmembrane 7 of the D2 dopamine receptor injected unilaterally into caudate nucleus of rats challenged with apomorphine resulted in rotational behavior, indicating D2 receptor blockade. No rotational behavior was seen with a similar peptide based on the 2 adrenergic receptor and the D2 transmembrane peptide did not affect the D1 dopamine receptor, indicating that the D2 receptor-derived peptide had a specific effect. The intravenous administration of a transmembrane peptide derived from the ₁-adrenergic receptor resulted in lowered arterial blood pressure and injection of a ₁-adrenergic receptor peptide resulted in decreased heart rate. Injection of a V2 vasopressin receptor-derived transmembrane peptide resulted in increased urine output, suggesting antagonism of the effects of vasopressin. Finally, dopamine release in rat brain after cocaine administration was blocked by a transmembrane peptide based on the dopamine transporter. Circular dichroism spectroscopy of the peptides revealed -helical structure similar to that of native transmembrane domains. Thus, transmembrane peptides can disrupt membrane proteins in vivo likely by competing with native transmembrane domains. The disruption of the hydrophobic core architecture of membrane proteins represents a novel mechanism of achieving functional inhibition that may be possible to exploit in developing novel therapeutics.

The dopamine transporter (DAT), like all catecholamine transporters, belongs to a large family of plasma membrane transporters that also includes carriers for GABA, glycine, proline, taurine, and betaine (Torres et al., 2003). Although no X-ray crystallographic or high-resolution structural information is available for the topological assignments of these transporters, membrane neurotransmitter transporters have been shown to contain 12 TM domains with intracellular amino and carboxyl termini (Chen and Reith, 2000). Like the GPCRs and as expected for a multiple membrane-spanning helix protein, the relative alignment of TM domains is believed to be specific and is critical for the overall three dimensional structure of the transporter and therefore its proper function (Nelson, 1998).

Peptides as protein or protein segment mimetics are frequently used in attempts to emulate known peptide/protein ligands (Kieber-Emmons et al., 1997) such as in the case of somatostatin (Freda, 2002) and gonadotropin-releasing hormone analogs (Kiesel et al., 2002). However, peptides mimicking proteins or protein segments have also been extensively and successfully used as "decoys" that interfere with or imitate the normal interactions of the protein that is being mimicked (Okamoto et al., 1991; Pan et al., 1997; Tanaka et al., 1998; da Silva Tatley et al., 2003). We have shown that in vitro incubation of a peptide derived from TM 6 of the D1 dopamine receptor with membranes from cells heterologously expressing the D1 dopamine receptor resulted in disrupted receptor function (George et al., 1998). The D1-TM6 peptide became inserted into the hydrophobic core of the D1 receptor and disrupted the ligand binding site, leading to attenuated ligand binding and signal transduction. Similarly, the use of TM peptides in vitro has been shown to impair 2-adrenergic (Hebert et al., 1996) and CCR5 (Tarasova et al., 1999) chemokine receptor functions. Therefore, the interhelical associations of GPCRs seem to be disturbed by exogenous peptides containing a TM sequence derived from the receptor itself as these peptides compete for the sites of interaction of the native TM domains, leading to a disruption of the receptor structure.

In this investigation, we examined whether the administration of TM peptides in vivo could attenuate receptor-mediated signal transduction and thereby elicit a behavioral or physiological response. Taking advantage of several well studied physiological functions known to be linked directly to the function of specific GPCRs, we examined TM peptides derived from the D2 dopamine, ₁-adrenergic, ₁-adrenergic, and V2 vasopressin receptors. In each case, the TM peptide disrupted the function of the receptor from which it was derived, suggesting that these peptides may represent a novel type of antagonist. We also attempted to determine whether this strategy for receptor disruption could be applied to another multiple membrane-spanning helix protein, the DAT. When a peptide derived from a TM domain of the DAT was administered to disrupt transporter function, blockade of cocaine-induced dopamine release was observed. Given the degree of specificity involved with interactions between TM domains in an integral membrane protein, our study indicates that there is great potential for the use of TM peptides or similar disrupters of hydrophobic core architecture as therapeutic agents.

	Materials and Methods

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Peptides. Peptides were synthesized using standard solid state methodology by Quality Control Biochemicals (Hopkinton, MA) (unless indicated otherwise). For experiments, 5 mg of peptide was dissolved in 100 µl of dimethyl sulfoxide and 100 µl of 5% (w/v) digitonin and taken up to 1 ml with buffer consisting of 100 mM NaCl, 10 mM Tris-HCl (pH 7.4), and 2 mM EDTA. Peptide amino acid sequences and the human receptor/transporter and TM region from which they were derived were as follows: D2 dopamine receptor TM 7, LYSAFTWLGYVNSAVNPIIY-NH2 (synthesized by Chiron Mimotopes, San Diego, CA); D2 dopamine receptor TM 5, PAFVVYSSIVSFYVPFIVTL-NH2; V2 vasopressin receptor TM 7, LMLLASLNSCTNPWIY-NH2; dopamine transporter TM 12, ALGWIIATS-NH2; GABA_A receptor subunit TM 1, GIFNLVYW-NH2; _1A-adrenergic receptor TM 7, GVFKVIFWLGYFNSCVNPL-NH2; ₁-adrenergic receptor TM 7, LFVFFNWLGYANSAFNPIIY-NH2; and ₂-adrenergic receptor TM 6, GIIMGTFTLCWLPFFIVNI VHCOOH

Circular Dichroism Spectroscopy of TM Peptides. Circular dichroism spectroscopy was performed using a 720 spectropolarimeter (Jasco, Tokyo, Japan) equipped with a variable temperature control unit (Thermo NESLAB, Portsmouth, NH). Cell path length was 0.1 cm, scan speed was 50 nm/min, resolution was 0.2 nm, and bandwidth was 1.0 nm. Peptides were dissolved as described above to a concentration and then diluted in 50 mM SDS, 0 to 100 mM NaCl, 10 mM Tris-HCl (pH 7.4), and 2 mM EDTA buffer to a concentration of 40 µM. Four scans were accumulated for each run; the spectra shown represent the smoothed mean of three separate trials for each experiment. Spectra at 37°C were recorded 2 min after this temperature was reached.

D2 Dopamine Receptor Expression and Radioligand Binding. The D2 dopamine receptor was expressed in Sf9 cells, and receptor binding was carried out on membranes as described previously (Ng et al., 1994), using [³H]spiperone (400 pM) with (+)-butaclamol (1 µM) to define specific binding.

Photoaffinity Labeling after Peptide Treatment. Membranes prepared from D2 receptor-expressing Sf9 cells were incubated for 30 min at 37°C with 1 µg/ml TM 7 peptide or peptide vehicle. After peptide treatment, the membranes were washed with phosphate-buffered saline and incubated for 1.5 h at room temperature with the photoaffinity label [¹²⁵I]4-azido-5-iodo-nemonapride (1 nM). This procedure and the synthesis of the photoaffinity compound have been described previously (Lee et al., 2000). Specific binding was defined by coincubation with 1 µM (+)-butaclamol. The possibility that D2-TM7 peptide may be labeled with [¹²⁵I]4-azido-5-iodonemonapride was excluded by incubating the photoaffinity agent with the peptide alone and subjecting the mixture to a high-percentage acrylamide gel electrophoresis. No crosslinking of the photoaffinity agent to the peptide was detected (data not shown).

Animal Welfare. All procedures involving animal subjects strictly adhered to guidelines and policies of the University of Toronto's Animal Use Protocol.

Peptide Injection in Rat Brain. Male Wistar rats (300-350 g) were anesthetized (66 mg/kg ketamine, 3 mg/kg acepromazine, and 22 mg/kg i.p. pentobarbital) for chronic stereotaxic implantation. A unilateral stainless steel guide cannula (22 gauge) was placed into the center of the left caudate putamen (anterior, +1.5; lateral, -2.2; vertical, -5.0) (Paxinos and Watson, 1982) to allow peptide or vehicle injection. Alternatively, bilateral stainless steel guide cannulae (22 gauge) were stereotaxically placed into the center of left and right caudate putamen for simultaneous drug and vehicle injections. The guide cannula was kept patent by a stylet (Plastics One, Inc., Roanoke, VA) terminating 0.5 mm below the guide tip, located 2 mm above the point of injection. The rats were allowed 1 wk of postoperative recovery before experimental use. The stylets were withdrawn and injections (peptide or vehicle) made into conscious rats using a 28-gauge internal cannula connected by PE-50 polyethylene tubing to a gastight Hamilton syringe. For unilateral operated animals, a total of 2- to 3-µl volume of peptide in vehicle or vehicle alone was injected into the left striatum, followed 15 min later by a subcutaneous injection of 0.25 mg/kg apomorphine. Rats with bilateral cannulae were administered peptide into the left striatum and vehicle into the right striatum simultaneously, followed 15 min later by a subcutaneous injection of 0.25 mg/kg apomorphine.

Ipsilateral Rotation Characterization. The intensity of the behavioral response was assessed every 2 to 5 min postinjection. Asymmetry (ipsilateral to the side of peptide or vehicle injections) was scored on the 0-3 system. Animals showing an ability to move in right and left directions were not categorized as circling. However, an ability to circle in one direction (asymmetric body posture) was scored on a 0-3 response according to observations in the open field and to the lifting of the tail. The criteria that met the 0-3 scores were as follows: 0, no asymmetry, response of animals the same as untreated rats; 1, a distinct tendency for animals to move in one direction when handled but still capable of movement in either direction; 2, spontaneous movements in one direction, a twisting of the body in this direction, exaggerated when handled, with inability to move in opposite direction; and 3, a marked and intense twisting of the body in one direction, active circling movements when disturbed, the animal being unable to move in the opposite direction.

Autoradiography of Ligand Binding in Rat Brain. Male Wistar rats implanted stereotactically with a cannula in the cerebroventricle were injected with D2-TM7 peptide (10 µg) or vehicle and were sacrificed 30 min later. The brains were rapidly resected, frozen on dry ice, and coronal sections (10 µm) through the caudate putamen (1.4 mm rostral to bregma) and nucleus accumbens (2.2 mm rostral to bregma) were obtained using a cryostat and thaw-mounted onto gelatin-coated slides. For the assay, the sections were air-dried for 30 min and then preincubated with buffer [50 mM Tris-HCl (pH 7.4), 5 mM EDTA, 2 mM CaCl₂, 1 mM MgCl₂, 5 mM KCl, and 120 mM NaCl] and then incubated with [³H]nemonapride (0.492 nM) to label D2 dopamine receptors or [³H]SCH 23390 (1 nM) to label D1 dopamine receptors. Nonspecific binding was defined by incubation with 1 µM (+)-butaclamol. Rimcazole (1 µM), a selective -receptor antagonist was added to the sections incubated with [³H]nemonapride to prevent its binding to -receptors. After incubation for 90 min, the slides were washed in ice-cold buffer twice, dipped into ice-cold deionized water to eliminate salts, dried, and exposed to Hyperfilm (Amersham Biosciences Inc., Piscataway, NJ) for 21 days. The autoradiographs were subjected to densitometric analysis (MCID; Imaging Research, St. Catherines, ON, Canada) and using radioisotopic standards, receptor densities were determined.

Cardiovascular Effects of TM Peptide. Male Wistar rats (250-270 g) were deprived of food for 12 h before surgery, but received water ad libitum. For vascular access, rats were premedicated with atropine, anesthetized with halothane, and the right carotid artery and the left jugular vein were cannulated with PE-50 catheters. The arterial line was used for blood pressure recording, and the jugular vein was used for injection of drugs. The animals were allowed to recover for a minimum of 3 h, so that the experiments were conducted in awake and nonrestrained animals. The baseline mean arterial pressure and heart rate values were based on 30 consecutive minutes of stable readings. All drugs and vehicle were administered in a volume of 200 µl.

Urine Output after Injection of a V2 Vasopressin Receptor TM Peptide. Male Wistar rats (250-270 g) were anesthetized with halothane, and the aorta and bladder were cannulated with PE-50 polyethylene catheters. The rats were unconscious and received a constant saline infusion before and during urine collection. Measurement of urine output volume started after urine flow stabilized. Urine was collected for 15 min before injection of the peptide and continued every 15 min until the conclusion of the experiment. A peptide derived from TM 7 of the V2 vasopressin receptor was injected 75 min after the injection of the vehicle.

Microdialysis Measurement of Dopamine. Rats were implanted with intracerebral guide cannulae (Plastics One, Inc.) for the microdialysis probes (Carnegie Medicin, Stockholm, Sweden) under anesthesia as described above. The cannula was inserted into the medial nucleus accumbens (shell region) (coordinates relative to bregma: A, 1.5; L, 1.3; V, 7.6) or into the center of the left caudate putamen (A, +1.5; L, -2.2; V; -5.0), and rats were allowed to recover for at least 5 days. On the day of the experiment, the guide cannula was replaced by the dialysis probe (2 mm, CMA/12; Carnegie Medicin) and perfused with artificial cerebral spinal fluid (145 mM NaCl, 2.7 mM KCl, 1.2 mM CaCl₂·2H₂O, 1.0 mM MgCl₂, 2 mM Na₂HPO₄, 0.2 mM ascorbic acid, pH 7.4) at a rate of 1 µl/min. Perfusion in the awake, unrestrained, and mobile animals was continued for 3 to 4 h until the basal efflux of dopamine and its metabolites (homovanillic acid and 3,4-dihydroxyphenylacetic acid) were stable for three consecutive measurements. The animals were then injected with 5 mg/kg i.p. cocaine. Twelve minutes after i.c.v. injection with 100 or 300 µg of DAT TM 12 peptide (n = 3 at each dose tested) or vehicle 5 µl (n = 8), dialysate was collected over 30-min periods and injected directly into a high-performance liquid chromatography system equipped with a Biophase ODS column (5 µm, 4.6 x 250 mm), Waters 590 pump (Waters, Milford, MA) with U6K injector and ESA 5100A electrochemical detector with model 5011 analytical cell for measurement of dopamine, 3,4-dihydroxyphenylacetic acid, and homovanillic acid. The mobile phase consisted of 50 mM sodium phosphate monobasic, 0.5 mM EDTA, 1.8 mM sodium octyl sulfonate, and 14% methanol, with pH adjusted to 3.50 with phosphoric acid. Sensitivity of dopamine detection was 2 pg. The percentage recovery of dopamine through the dialysis cannula was calculated each time.

	Results

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The TM domains of GPCRs are predicted to exist as -helices within the membrane (Liu and Deber, 1998), which in turn promotes packing and assembly to align the TM domains in a highly organized structure (Palczewski et al., 2000). To determine the structure of the present synthetic TM-based peptides, their circular dichroism (CD) spectra were recorded in the membrane-mimetic environment of sodium dodecylsulfate micelles, as shown in Fig. 1. CD spectra of both the D2-TM7 and the 2-TM6 peptides at 25°C and 37°C displayed significant -helical character, exhibiting minima near 208 and 222 nm. The fact that both peptides were -helical in micelles under physiological salt and temperature conditions indicates their conformational potential to interact with other native -helices. Furthermore, structural correspondence has been observed for nonpolar peptide segments of intact proteins, both in their ability to retain -helical structure (Voss et al., 1993; Myers et al., 1997b; Wang and Deber, 2000) and to stabilize sequence-dependent helix-helix interactions within membranes (Myers et al., 1997a). It is highly conceivable, therefore, that the synthetic TM peptides acted in vivo by mimicking the native TM domain morphology.

View larger version (23K): [in this window] [in a new window]   Fig. 1. CD spectra of the synthetic D2-TM7 and 2-TM6 peptides at varying temperatures. Each peptide (40 µM) was solubilized as described under Materials and Methods and diluted in buffer containing 50 mM SDS, 10 mM Tris-HCl (pH 7.4), and 2 mM EDTA, with 100 mM NaCl. The result shown is representative of the three replicates of this experiment that were performed.

In an attempt to disrupt the intramolecular interactions among TM domains, a synthetic peptide derived from TM domain 7 of the D2 dopamine receptor was synthesized. Because the TM domains of a GPCR are arranged in a circular formation, with the TM domains 1 and 7 adjacent to each other (Suryanarayana et al., 1992; Strader et al., 1994), we hypothesized that a peptide based on one of these terminal TM domains would be the most effective at interacting with the receptor. The D2-TM7 peptide inhibited binding of the dopamine antagonist [³H]spiperone to membranes from cells expressing D2 receptors in a dose-dependent manner (Fig. 2A). A peptide derived from the TM 5 of the D2 receptor also decreased binding, but to a lesser extent. Peptides derived from the TM 7 domain of the vasopressin V2 receptor, the TM 12 region of the dopamine transporter and TM 1 region of the GABA_A receptor subunit had negligible effect on [³H]spiperone binding to the D2 dopamine receptor. The D2-TM7 peptide also inhibited binding of the agonist [³H]quinpirole to the D2 dopamine receptor (Fig. 2B) in a manner similar to a classical dopamine antagonist (Fig. 2C), and prevented binding and incorporation of the D2-selective photoaffinity ligand [¹²⁵I]azidonemonapride to the D2 receptor species (Fig. 2D). These results are consistent with a previous in vitro study that showed that a peptide derived from a TM domain of the D1 dopamine receptor acted as an irreversible inhibitor of D1 receptor function (George et al., 1998). Therefore, in vitro, treatment of the D2 dopamine receptor with the D2-TM7 peptide resulted in the disruption of ligand binding to the receptor.

View larger version (17K): [in this window] [in a new window]   Fig. 2. Effect of TM peptides on radioligand and photoaffinity ligand interaction with the D2 dopamine receptor. A, inhibition by transmembrane domain peptides of [3H]spiperone binding to the dopamine D2 receptor expressed in Sf9 cells. The peptides correspond to the sequences, respectively, of the dopamine D2 receptor TM 7 (D2-TM7) and TM 5 (D2-TM5), the V2 vasopressin receptor TM 7 (V2-TM7), the GABAA receptor subunit TM 1 (GABA-TM), and the dopamine transporter TM 12 (DAT-TM12). The effect of the peptides is shown as the mean ± S.E.M. percentage of the total [3H]spiperone binding from three independent experiments. Statistically significant differences, as determined by t test, are noted by asterisks. Dose-dependent competition for [3H]quinpirole binding to the D2 dopamine receptor expressed in Sf9 cells by D2-TM7 peptide (B) and spiperone (C). The data shown are representative of three replicates. D, effect of D2-TM7 peptide on [125I]azidonemonapride labeling of D2 receptors expressed in Sf9 cells. Photoaffinity labeling of D2 receptors monomers (48 kDa), dimers (98 kDa), and tetramers (200 kDa) was visualized (lane 1) that was prevented by prior incubation with the D2-TM7 peptide (1 µg/ml) (lane 2) or (+)-butaclamol (1 µM) (lane 3). The autoradiogram shown is representative of the five identical photoaffinity labeling experiments performed.

Receptor autoradiography of rat brain after i.c.v. injection of the D2-TM7 peptide revealed reduced ligand binding to the D2 dopamine receptor (Fig. 3, A and B). Blockade of D2 dopamine receptor binding was analogous to that achieved by the classical antagonist (+)-butaclamol (Fig. 3C). Notably, when ligand binding to the D1 dopamine receptor was examined in animals treated with D2-TM7 peptide, there was no effect (Fig. 3, D-F), indicating that the peptide had a specific and selective antagonist effect in vivo on the ligand binding capability of the D2 receptor.

View larger version (102K): [in this window] [in a new window]   Fig. 3. Autoradiography of D1 and D2 dopamine receptors in coronal sections of rat brain after administration of D2-TM7 peptide or vehicle i.c.v. Rat brain sections showing binding of [3H]nemonapride (0.492 nM) to D2 dopamine receptors (A-C) or [3H]SCH 23390 (1 nM) to D1 dopamine receptors (D-F). Sections are from rats treated with vehicle (A and D) or the D2-TM7 peptide (B and E). Nonspecific binding was defined by incubation of the vehicle-treated sections with 1 µM (+)-butaclamol (C and F). CP, caudate putamen; Acb, nucleus accumbens; OT, olfactory tubercle. The data shown are representative of six replicates.

The function of the D2-TM7 peptide was tested in vivo for D2 receptor antagonist activity using an animal model for striatal function, established for screening dopamine receptor antagonists (Costall et al., 1983). Awake, freely mobile rats bearing stereotactically guided cannulae were injected into the left caudate nucleus with peptides derived from TM 7 of the D2 receptor or TM 6 of the ₂-adrenergic receptor. The injection of either peptide did not have an effect on the locomotion of the animal. However, after subcutaneous challenge with the dopamine analog apomorphine 15 min later, an ipsilateral asymmetric body rotation in rats administered D2-TM7 peptide was observed, achieving a behavioral response score that was not observed in rats injected with the ₂-adrenergic receptor TM 6 peptide (Fig. 4A). Similar ipsilateral asymmetric rotation was also documented in rats after bilateral intracaudate administration of D2-TM7 peptide and vehicle simultaneously into left and right caudate nucleus, respectively (Fig. 4B). These results indicate that the D2-TM7 peptide acted in the animal model as a functional D2 receptor antagonist.

View larger version (13K): [in this window] [in a new window]   Fig. 4. Rotational activity induced by administration of TM peptides into the caudate nucleus of rats. A, apomorphine-induced asymmetric body rotation after unilateral (left side) administration of D2-TM7 peptide or 2-TM6 peptide into caudate nucleus. The duration and extent (score) of the asymmetric body response after injection of D2-TM7 peptide (15 ng/3 µl) and 2-TM6 peptide, (15 ng/3 µl) are shown. B, apomorphine-induced asymmetric body rotation after bilateral simultaneous administration into caudate nucleus of D2-TM7 peptide (left side) and vehicle (right side). The duration and extent of asymmetric body response of a rat with bilateral cannulae after injection of D2-TM7 peptide (15 ng/3 µl) and vehicle (3 µl) concurrently are shown. The data shown are representative of three replicates.

To test the universal applicability of our hypothesis, the actions of peptides designed based on the sequences of other GPCRs were tested for effects to inhibit the activity of selected receptors. A peptide based on the TM 7 of the _1A-adrenergic receptor, if an effective -adrenergic antagonist, would be hypothesized to lower arterial blood pressure. Administration of an _1A-adrenergic receptor TM 7 peptide i.v. into rats implanted with carotid cannulae resulted in a reduction of systolic and diastolic blood pressure, with a concomitant rise in the heart rate (Fig. 5A). In comparison, the _1A-adrenergic receptor antagonist prazosin also lowered blood pressure and elevated heart rate (Fig. 5B). The effect of the -adrenergic agonist phenylephrine to elevate blood pressure was attenuated when administered after the _1A-adrenergic receptor TM 7 peptide (Fig. 5, C and D). In addition, a peptide based on the TM 7 of the ₁-adrenergic receptor was predicted to lower heart rate and was tested for effects on spontaneous cardiac activity and blood pressure in rats. With this peptide, a short-lived but marked slowing of heart rate was revealed with a selective drop in diastolic blood pressure (Fig. 5F).

View larger version (34K): [in this window] [in a new window]   Fig. 5. Arterial pressure and heart rate recording from carotid artery cannulated rats after administration of TM peptides based on adrenergic receptors. Panels show effects of the 1-adrenergic receptor TM 7 peptide (A) and prazosin (B). Effects of phenylephrine (C), phenylephrine after 1A-adrenergic receptor TM 7 peptide (D), vehicle (E), and 1-adrenergic receptor TM 7 peptide (F) are shown. Peptide or drug was administered at time 0. The data shown is representative of three replicates.

It was then hypothesized that a peptide derived from TM 7 of the V2 vasopressin receptor could block the actions of vasopressin (antidiuretic hormone) on the V2 vasopressin receptor and therefore act as a diuretic. Injection of peptide vehicle through an aortic cannula in a rat resulted in a slight increase in urine output compared with measurements made before injection (Fig. 6). However, the in vivo administration of V2-TM7 peptide resulted in diuresis much greater than that seen with vehicle injection. These observations suggested that the V2 vasopressin receptor TM 7 peptide was disrupting the antidiuretic function of the V2 vasopressin receptor.

View larger version (14K): [in this window] [in a new window]   Fig. 6. Urine output for bladder-cannulated rat after aortic injection of peptide vehicle and a peptide derived from TM domain 7 of the V2 vasopressin receptor. Injection of the peptide vehicle was performed 15 min after the initial urine output measurement and the time of the injection was considered time 0. The peptide was administered 75 min after the vehicle. Injection times are shown by arrows. The data shown are representative of three replicates.

To determine whether the principle of TM domain interference with receptor function could be extrapolated to other membrane proteins with multiple membrane-spanning domains, a peptide based on one of the terminal TM domains of the DAT, TM 12, was synthesized. The peptide was used in an experiment wherein cocaine-induced dopamine release from nucleus accumbens and caudate nucleus of live rats could be reliably measured by a stereotactically implanted microdialysis probe. It was demonstrated that pretreatment with the 100 or 300 µg of DAT TM 12 peptide resulted in complete suppression of the cocaine-induced dopamine release detected in vehicle treated control animals (Fig. 7), indicating functional antagonism of cocaine action. It was not clear whether this blockade was due to an inhibition of cocaine binding, a disruption of transporter activity, or a combination of both actions. Interestingly, administration of 100 µg of the DAT TM 12 peptide alone (Fig. 7A) had no effect on basal dopamine release from caudate nucleus or nucleus accumbens. However, administration of 300 µg of the DAT TM 12 peptide alone produced a gradual increase in basal dopamine release (Fig. 7B) as well as a similar increase in basal DOPAC and HVA levels (data not shown). Therefore, it seemed that the peptide interfered with transporter activity and did not solely inhibit cocaine binding.

View larger version (27K): [in this window] [in a new window]   Fig. 7. In vivo microdialysis measurement of dopamine release after cocaine administration. Release of dopamine from shell of nucleus accumbens was monitored after cocaine administration with and without pretreatment with 100 µg (A) or 300 µg (B) of DAT TM 12 peptide. Cocaine (5 mg/kg) was injected i.p. at time 0, and vehicle or DAT TM 12 peptide was injected i.c.v. at -12 min. The data shown are representative of three replicates.

	Discussion

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In this report, we postulated that peptides derived from the TM domains of GPCRs and monoamine transporters, when administered in vivo, may disrupt the function of the protein from which they are derived. To test this hypothesis, we used well characterized behavioral and physiological endpoints and tested several GPCR-derived TM peptides and a TM peptide based on the DAT. We demonstrated that, when a peptide derived from a transmembrane domain of the D2 dopamine receptor was injected into caudate nucleus of conscious, freely mobile rats and challenged with apomorphine, rotational behavior resulted, indicating D2 receptor blockade. This effect was not seen in rats injected with a similar peptide derived from the ₂-adrenergic receptor. The i.v. administration of a TM peptide derived from the ₁-adrenergic receptor resulted in lowered arterial blood pressure and injection of a ₁-adrenergic receptor peptide resulted in decreased heart rate, suggesting that these peptides behaved as antagonists for the ₁- and ₁-adrenergic receptors, respectively. We then showed in vivo antagonism of vasopressin activation of the V2 vasopressin receptor by a V2 receptor-derived TM peptide. Finally, we demonstrated that dopamine release from caudate nucleus and nucleus accumbens in rats after cocaine administration was blocked by the DAT-based TM peptide.

This study provides evidence for the first time in vivo that peptides derived from the TM domains of GPCRs and monoamine transporters may, by a novel mechanism of functional disruption, represent an atypical type of antagonist for these proteins. We propose that the mechanism of action of the TM-based peptides involves their specific interactions with complementary TM domains/segments within the integral membrane protein, thereby competing for the normal intramolecular interactions of the native TM domains (Baldwin et al., 1997). As we have shown by circular dichroism spectroscopy, exogenous TM peptides retain -helical properties similar to that proposed for native transmembrane domains. Not surprisingly therefore, TM domain-derived peptides have been shown to be good predictors of helix-helix interactions in membrane proteins (Wang and Deber, 2000; Melnyk et al., 2001; Therien and Deber, 2002). Selective stabilization of helices by neighboring helices, with significant, but not unbreakable, anchoring forces between particular adjacent helices has been demonstrated for rhodopsin (Palczewski et al., 2000) and the structurally analogous membrane protein bacteriorhodopsin (Oesterhelt et al., 2000). These interactions contribute to the proper three-dimensional folding and conformation of the protein and in the creation of the ligand binding pocket, often deep within the TM core. We predict that integral membrane proteins having one or more TM domains will be susceptible to disruption of their structure and therefore function, by peptides having the amino acid sequence of a TM segment that participates in forming the ligand-binding crevice. An antagonist peptide having the amino acid sequence of the TM domain of a particular integral membrane protein would likely show sequence-dependent specificity for that protein and, as demonstrated in the present work, may not interfere with the function of related proteins. Admittedly, there are obvious problems from the clinical viewpoint in using peptides such as the ones that were studied in this report as therapeutic agents. For example, drug delivery to the site of action and the longevity/stability present major challenges in developing effective peptide drugs (Bickel et al., 2001; Torchilin and Lukyanov, 2003). Nevertheless, the potential advantages in being highly selective to a particular class or subtype of receptor (or proteins with multiple membrane-spanning helices) obviously make the targeting of intramembrane interactions very intriguing.

In conclusion, we and others have previously shown disruption of GPCR function in vitro by peptides derived from TM domains of these receptors. Now, in this study, we have shown by CD spectra that TM peptides based on TM domains of GPCRs, when solubilized in a membrane-mimetic environment, have helical structures like native TM domains. We demonstrated that these TM peptides can elicit behavioral and physiological effects in vivo by selectively inhibiting the function of the receptor from which it was derived. Furthermore, we have expanded the concept and shown that other multiple TM domain proteins such as the DAT can also be disrupted in vivo by a peptide based on one of the TM helices of the protein. Our findings may provide a key to developing a novel category of therapeutic agents that disrupts the hydrophobic core architecture of integral membrane proteins and may be of benefit in the rational design of drugs targeting these proteins.

	Acknowledgements

 
We thank Victor Saldivia, Angeliki Kouvelas, and Melissa Tolis for technical assistance.

	Footnotes

 
DOI: 10.1124/jpet.103.053843.

This work was supported by grants from the Canadian Institutes of Health Research, the National Institute on Drug Abuse, and the Natural Sciences and Engineering Research Council of Canada, and by a Canada Research Chair to S.R.G.

ABBREVIATIONS: GPCR, G protein-coupled receptor; TM, transmembrane; DAT, dopamine transporter; PE, polyethylene; CD, circular dichroism; SCH 23390, R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1-H-3-benzazepine.

¹ Current address: Amgen, One Amgen Center Dr., Thousand Oaks, CA 91320-1799.

Address correspondence to: Dr. Susan R. George, Rm. 4358, Medical Sciences Bldg., 1 King's College Circle, University of Toronto, Toronto, ON M5S 1A8 Canada. E-mail address: s.george{at}utoronto.ca

	References

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Baldwin JM (1993) The probable arrangement of the helices in G protein-coupled receptors. EMBO (Eur Mol Biol Organ) J 12: 1693-1703.[Medline]
Baldwin JM, Schertler GF, and Unger VM (1997) An alpha-carbon template for the transmembrane helices in the rhodopsin family of G-protein-coupled receptors. J Mol Biol 272: 144-164.[CrossRef][Medline]
Ballesteros JA, Shi L, and Javitch JA (2001) Structural mimicry in G protein-coupled receptors: implications of the high-resolution structure of rhodopsin for structure-function analysis of rhodopsin-like receptors. Mol Pharmacol 60: 1-19.[Abstract/Free Full Text]
Bickel U, Yoshikawa T, and Pardridge WM (2001) Delivery of peptides and proteins through the blood-brain barrier. Adv Drug Deliv Rev 46: 247-279.[CrossRef][Medline]
Chen N and Reith MEA (2000) Structure and function of the dopamine transporter. Eur J Pharmacol 405: 329-339.[CrossRef][Medline]
Costall B, Kelly ME, and Naylor RJ (1983) The production of asymmetry and circling behaviour following unilateral, intrastriatal administration of neuroleptic agents: a comparison of abilities to antagonise striatal function. Eur J Pharmacol 96: 79-86.[CrossRef][Medline]
da Silva Tatley F, Aldwell FE, Dunbier AK, and Guilford PJ (2003) N-Terminal E-cadherin peptides act as decoy receptors for Listeria monocytogenes. Infect Immun 71: 1580-1583.[Abstract/Free Full Text]
Freda PU (2002) Somatostatin analogs in acromegaly. J Clin Endocrinol Metab 87: 3013-3018.[Free Full Text]
George SR, Lee SP, Varghese G, Zeman PR, Seeman P, Ng GYK, and O'Dowd BF (1998) A transmembrane domain-derived peptide inhibits D1 dopamine receptor function without affecting receptor oligomerization. J Biol Chem 273: 30244-30248.[Abstract/Free Full Text]
Hebert TE, Moffett S, Morello JP, Loisel TP, Bichet DG, Barret C, and Bouvier M (1996) A peptide derived from a beta2-adrenergic receptor transmembrane domain inhibits both receptor dimerization and activation. J Biol Chem 271: 16384-16392.[Abstract/Free Full Text]
Kieber-Emmons T, Murali R, and Greene MI (1997) Therapeutic peptides and peptidomimetics. Curr Opin Biotechnol 8: 435-441.[CrossRef][Medline]
Kiesel LA, Rody A, Greb RR, and Szilagyi A (2002) Clinical use of GnRH analogues. Clin Endocrinol 56: 677-687.[CrossRef][Medline]
Lee SP, O'Dowd BF, Ng GY, Varghese G, Akil H, Mansour A, Nguyen T, and George SR (2000) Inhibition of cell surface expression by mutant receptors demonstrates that D2 dopamine receptors exist as oligomers in the cell. Mol Pharmacol 58: 120-128.[Abstract/Free Full Text]
Liu LP and Deber CM (1998) Guidelines for membrane protein engineering derived from de novo designed model peptides. Biopolymers 47: 41-62.[CrossRef][Medline]
MacKenzie KR, Prestegard JH, and Engelman DM (1997) A transmembrane helix dimer: structure and implications. Science (Wash DC) 276: 131-133.[Abstract/Free Full Text]
Melnyk RA, Partridge AW, and Deber CM (2001) Retention of native-like oligomerization states in transmembrane segment peptides: application to the Escherichia coli aspartate receptor. Biochemistry 40: 11106-11113.[CrossRef][Medline]
Myers JK, Pace CN, and Scholtz JM (1997a) A direct comparison of helix propensity in proteins and peptides. Proc Natl Acad Sci USA 94: 2833-2837.[Abstract/Free Full Text]
Myers JK, Pace CN, and Scholtz JM (1997b) Helix propensities are identical in proteins and peptides. Biochemistry 36: 10923-10929.[CrossRef][Medline]
Nelson N (1998) The family of Na+/Cl neurotransmitter transporters. J Neurochem 71: 1785-1803.[Medline]
Ng GY, O'Dowd BF, Caron M, Dennis M, Brann MR, and George SR (1994) Phosphorylation and palmitoylation of the human D2L dopamine receptor in Sf9 cells. J Neurochem 63: 1589-1595.[Medline]
Oesterhelt F, Oesterhelt D, Pfeiffer M, Engel A, Gaub HE, and Muller DJ (2000) Unfolding pathways of individual bacteriorhodopsins. Science (Wash DC) 288: 143-146.[Abstract/Free Full Text]
Okamoto T, Murayama Y, Hayashi Y, Inagaki M, Ogata E, and Nishimoto I (1991) Identification of a Gs activator region of the beta 2-adrenergic receptor that is autoregulated via protein kinase A-dependent phosphorylation. Cell 67: 723-730.[CrossRef][Medline]
Palczewski K, Kumasaka T, Hori T, Behnke CA, Motoshima H, Fox BA, Le Trong I, Teller DC, Okada T, Stenkamp RE, et al. (2000) Crystal structure of rhodopsin: a G protein-coupled receptor. Science (Wash DC) 289: 739-745.[Abstract/Free Full Text]
Pan G, Ni J, Wei YF, Yu G, Gentz R, and Dixit VM (1997) An antagonist decoy receptor and a death domain-containing receptor for TRAIL. Science (Wash DC) 277: 815-818.[Abstract/Free Full Text]
Paxinos G and Watson C (1982) The Rat Brain in Stereotaxic Coordinates, Academic Press, New York.
Perlman JH, Colson AO, Wang W, Bence K, Osman R, and Gershengorn MC (1997) Interactions between conserved residues in transmembrane helices 1, 2 and 7 of the thyrotropin-releasing hormone receptor. J Biol Chem 272: 11937-11942.[Abstract/Free Full Text]
Scheer A, Fanelli F, Costa T, De Benedetti PG, and Cotecchia S (1996) Constitutively active mutants of the alpha 1B-adrenergic receptor: role of highly conserved polar amino acids in receptor activation. EMBO (Eur Mol Biol Organ) J 15: 3566-3578.[Medline]
Sealfon SC, Chi L, Ebersole BJ, Rodic V, Zhang D, Ballesteros JA, and Weinstein H (1995) Related contribution of specific helix 2 and 7 residues to conformational activation of the serotonin 5-HT2A receptor. J Biol Chem 270: 16683-16688.[Abstract/Free Full Text]
Shai Y (1995) Molecular recognition between membrane-spanning polypeptides. Trends Biochem Sci 20: 460-464.[CrossRef][Medline]
Sheikh SP, Vilardarga JP, Baranski TJ, Lichtarge O, Iiri T, Meng EC, Nissenson RA, and Bourne HR (1999) Similar structures and shared switch mechanisms of the beta2-adrenoceptor and the parathyroid hormone receptor. Zn(II) bridges between helices III and VI block activation. J Biol Chem 274: 17033-17041.[Abstract/Free Full Text]
Strader CD, Fong TM, Tota MR, Underwood D, and Dixon RA (1994) Structure and function of G protein-coupled receptors. Annu Rev Biochem 63: 101-132.[CrossRef][Medline]
Suryanarayana S, von Zastrow M, and Kobilka BK (1992) Identification of intramolecular interactions in adrenergic receptors. J Biol Chem 267: 21991-21994.[Abstract/Free Full Text]
Tanaka T, Kohno T, Kinoshita S, Mukai H, Itoh H, Ohya M, Miyazawa T, Higashijima T, and Wakamatsu K (1998) Alpha helix content of G protein alpha subunit is decreased upon activation by receptor mimetics. J Biol Chem 273: 3247-3252.[Abstract/Free Full Text]
Tarasova NI, Rice WG, and Michejda CJ (1999) Inhibition of G-protein-coupled receptor function by disruption of transmembrane domain interactions. J Biol Chem 274: 34911-34915.[Abstract/Free Full Text]
Therien AG and Deber CM (2002) Oligomerization of a peptide derived from the transmembrane region of the sodium pump gamma subunit: effect of the pathological mutation G41R. J Mol Biol 322: 583-590.[CrossRef][Medline]
Torchilin VP and Lukyanov AN (2003) Peptide and protein drug delivery to and into tumors: challenges and solutions. Drug Discov Today 8: 259-266.[CrossRef][Medline]
Torres GE, Carneiro A, Seamans K, Fiorentini C, Sweeney A, Yao W-D, and Caron MG (2003) Oligomerization and trafficking of the human dopamine transporter. Mutational analysis identifies critical domains important for the functional expression of the transporter. J Biol Chem 278: 2731-2739.[Abstract/Free Full Text]
Voss T, Wallner E, Czernilofsky AP, and Freissmuth M (1993) Amphipathic alpha-helical structure does not predict the ability of receptor-derived synthetic peptides to interact with guanine nucleotide-binding regulatory proteins. J Biol Chem 268: 4637-4642.[Abstract/Free Full Text]
Wang C and Deber CM (2000) Peptide mimics of the M13 coat protein transmembrane segment. Retention of helix-helix interaction motifs. J Biol Chem 275: 16155-16159.[Abstract/Free Full Text]

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