Characterization of [3H ]5-hydroxytryptamine and [3H ]spiperone binding sites in clathrin-coated vesicles from bovine brain

Brain Research 794 Ž1998. 291–298

Research report

Characterization of w3 Hx5-hydroxytryptamine and w3 Hxspiperone binding sites in clathrin-coated vesicles from bovine brain Kayoko Moroi a

a, )

, Naoko Ozaki b , Tomoko Kadota c , Ken Kadota

d

DiÕision of CardioÕascular Biology, Center for Biomedical Science, Chiba UniÕersity School of Medicine, Chiba, 260, Japan b Department of Pharmacology, Kanazawa Medical UniÕersity, Uchinada, Isikawa, 920-20, Japan c Department of Anatomy, Chiba UniÕersity School of Medicine, Chiba, 260, Japan d Chiba City Institute of Health and EnÕironment, Chiba, 261, Japan Accepted 3 March 1998

Abstract Coated vesicles prepared from bovine brain cerebral cortex exhibited w3 Hx5-hydroxytryptamine Ž5-HT, serotonin. and w3 Hxspiperone binding activities. The binding activities were localized in the inner core vesicles. Binding reached an equilibrium level by 30–45 min at 308C, and was reversed by the addition of 100 mM 5-HT for w3 Hx5-HT binding or 10 mM ketanserin for w3 Hxspiperone binding. The saturation binding experiments indicated a single class of binding sites for w3 Hx5-HT and w3 Hxspiperone with apparent K d values of 2.4 and 1.75 nM, respectively. The binding of w3 Hx5-HT was displaced by 5-HT and 8-hydroxy-2-Ždi-n-propylamino.-tetralin Ž8-OH-DPAT., but not by ketanserin. The binding of w3 Hxspiperone was displaced by spiperone and ketanserin but not by 5-HT or 8-OH-DPAT even at 1 mM. The coated vesicles were shown by immunoblotting assay to contain a-subunits of GTP-binding proteins, Ga s, Ga i2, Ga i3, Ga o and Ga qr11. Forskolin-stimulated adenylate cyclase activity in the coated vesicles was inhibited to 80% of the control level by 5-HT or 8-OH-DPAT. These results suggested that 5-HT1A and 5-HT2A receptors are present in bovine brain coated vesicles and that the 5-HT1A receptors are coupled to adenylate cyclase activity via GTP binding proteins. q 1998 Elsevier Science B.V. All rights reserved. Keywords: w3 Hx5-hydroxytryptamine binding; w3 Hxspiperone binding; Serotonin receptor, 5-HT1 and 5-HT2 receptor; Coated vesicle; Bovine brain

1. Introduction Clathrin-coated vesicles with a characteristic outer coat structure have been implicated in internalization of numerous macromolecules, growth factors and nutrients via receptor-mediated endocytosis. This type of endocytosis involves the formation of coated pits at the plasma membrane that develop into coated vesicles w30x. The coat structures are composed predominantly of clathrin heavy chains Ž180 kDa protein., three clathrin light chains Ž33–36 kDa protein. and a protein complex composed of 100, 50 and 16 kDa components termed assembly polypeptides, accessory proteins or adaptor proteins ŽAP2. w19x. Recent biochemical studies revealed that the AP2 complex plays an essential role in endocytosis: AP2 is able to bind to the cytoplasmic tail of membrane receptors and is likely to initiate the formation of clathrin-coated pits w7,42x. ) Corresponding author. Division of Cardiovascular Biology, Center for Biomedical Science, Chiba University School of Medicine, Inohana, 1-8-1, Chuo-ku, Chiba, 260, Japan. Fax: q81-43-226-2196.

0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 2 4 5 - 5

Agonist-induced desensitization is a common feature of G protein-coupled signal transduction systems. One type of receptor desensitization is characterized by phosphorylation of serine and threonine residues of the receptors by second messenger-dependent protein kinases, c-AMP-dependent protein kinase and protein kinase C ŽPKC., and by G protein-coupled receptor kinases ŽGRKs. w22x. The phosphorylated receptors consequently bind to cytosolic proteins referred to as arrestins, followed by internalization via dynamine-associated coated vesicle formation w11x. Recently, b 2-adrenergic receptors have been demonstrated to bind in vitro to clathrins through b-arrestins as adaptors w14x. This strongly suggests that the other seven-transmembrane receptors may be internalized via a similar pathway through clathrin-coated vesicles after agonist-induced phosphorylation. The neurotransmitter serotonin Ž5-hydroxytryptamine, 5-HT. has diverse psychological and physiological effects including the regulation of sleep, aggression, platelet aggregation and smooth muscle contraction. Serotonin exerts its effects by interaction with specific membrane receptors

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which have been classified into pharmacologically distinct subtypes w17x. The 5-HT receptors are rapidly desensitized by treatment with agonists, and phosphorylation of 5-HT receptors by PKC w36x and GRKs w28x has been reported to be one mechanism responsible for this effect. The presence of neurotransmitter receptors such as opiate w3x, a 2-adrenergic w26x, b-adrenergic w8x, muscarinic acetylcholine w41x, adenosine A 1 w13x, glutamate w25x, dopamine D1 and D 2 w29x receptors in coated vesicles from bovine brain has been reported. Some of these receptors, i.e., adenosine A 1 w13,37x, dopamine D 1 and D 2 w29x and glutamate w25x receptors, are associated with G proteins and adenylate cyclase activity. In the present study, we determined whether 5-HT receptors are present in coated vesicles from the bovine cerebral cortex using w3 Hx5-HT and w3 Hxspiperone as ligands. The results showed that the w3 Hx5-HT and w3 Hxspiperone binding sites in the coated vesicles have the properties of 5-HT1A and 5-HT2A receptors, respectively, and that the 5-HT1A receptors are associated with adenylate cyclase activity via Giro-proteins.

2. Materials and methods

in an equal volume Žvrw. of 50 mM 2-Ž N-morpholino. ethane–sulfonic acid buffer ŽpH 6.0. containing 1 mM EGTA, 0.5 mM MgCl 2 and 0.02% NAN3 ŽMES buffer.. The homogenate was centrifuged at 20,000 = g for 30 min followed by sedimentation at 55,000 = g for 1 h. The resulting pellets suspended in MES buffer were centrifuged through a 30–50% continuous sucrose density gradient at 55,000 = g for 15 h. A band at 38–45% sucrose was subjected to second centrifugation with a continuous sucrose density gradient of 5–30% at 100,000 = g for 1 h. The band at 14–20% sucrose was collected and precipitated by centrifugation as the purified coated vesicle preparation. The samples were suspended in 50 mM MES buffer and stored at y808C until use. To prepare separate decoated vesicles and coat structure fractions, the coated vesicles were incubated with an equal volume of MES buffer containing 1 M Tris–HCl, pH 8.0, at 258C for 2 h. After incubation, the mixture was centrifuged at 100,000 = g for 1 h to separate the pellet Ždecoated vesicles; core. and supernatant Žcoat fraction.. The supernatant was dialyzed against 50 mM MES buffer overnight at 48C and centrifuged at 100,000 = g for 1 h to recover the reconstructed coat structures. Protein concentration was determined by the method of Lowry et al. w23x using bovine serum albumin as a standard.

2.1. Materials w3 Hx-5-Hydroxytryptamine Ž5-HT. Ž30 Ci mmoly1 ., w3 Hxspiperone Ž27.5 Ci mmoly1 ., 8-hydroxy-2-Ždi-n-propylamino.-tetralin Ž8-OH-DPAT,142.9 Ci mmoly1 ., anti-G protein antibodies ŽGa s, Ga o, Ga qr11., and chemiluminescence detection reagent were obtained from Du PontNEN ŽBoston, MA, USA.. Cyclic-AMP detection kit was from Yamasa ŽChyoshi, Chiba, Japan.. Anti-G protein antibodies ŽGa i1, Ga i2 and Ga i3. were from Wako Pure Chemicals ŽOsaka, Japan.. 5-HT creatinine sulfate, guanosine triphosphate ŽGTP., guanyl-5yl imidodiphosphate ŽGppŽNH.p., adenosine triphosphate ŽATP., EGTA, pargyline, creatine kinase, creatine phosphate, forskolin were purchased from Sigma ŽSt. Louis, USA.. 8-Hydroxy-2-Ždin-propylamino.-tetralin Ž8-OH-DPAT. and 4-wŽ3-butoxy4-methoxyphenyl.-methylx-2-imidazolidinone ŽRo20-1724. were from Research Biochemicals ŽBoston, MA, USA.. The following reagents were kind gifts from the sources shown; Methysergide ŽSandoz, Switzerland., Ketanserin ŽJanssen-Kyowa, Tokyo, Japan., Mianserin ŽJapan Organo, Tokyo, Japan. and Spiperone ŽEizai, Tokyo, Japan.. All other reagents used were of analytical grade. 2.2. Isolation of coated Õesicles Coated vesicles were prepared from fresh bovine brains as previously described w26x. Briefly, about 800 g of the superficial layer of the frontal and parietal cortices was carefully scraped off with a razor blade and homogenized

2.3. Binding assay Radiolabeled ligands, w3 Hx5-HT and w3 Hxspiperone, were used in the binding assays to identify 5-HT1 and 5-HT2A receptors, respectively w31x. In a standard assay, coated vesicles Ž200–300 mg protein. were incubated with 4 nM w3 Hx5-HT or 1 nM w3 Hxspiperone in 50 mM Tris–HCl buffer containing 5.7 mM ascorbic acid, 4 mM CaCl 2 and 10 mM pargyline ŽpH 7.4. in a total volume of 0.5 ml at 308C for 30 min. The reaction was started by adding radiolabeled ligands and terminated by adding 3 ml of ice-cold buffer and rapid filtration through Whatman GFrC filters. The filters were immediately washed three times with 3 ml of ice-cold buffer. The radioactivity on the filters was counted in a liquid scintillation counter. Specific binding was determined as the difference between the total binding and that in the presence of 100 mM 5-HT for w3 Hx5-HT or 10 mM ketanserin for w3 Hxspiperone. Ketanserin, a specific 5-HT2A antagonist w21x, was used to define the 5-HT2A receptors. In w3 Hx5-HT binding assay for 5-HT1 receptors, 0.1 mM ketanserin was added to the incubation medium to eliminate 5-HT2A binding sites. To specify the 5-HT1 and 5-HT2 receptor subtypes, various agonists and antagonists were used in displacement experiments, and the reagents were added to the assay medium 5 min before w3 Hx5-HT or w3 Hxspiperone. For Scatchard plots, the coated vesicles were incubated with various concentrations of w3 Hx5-HT or w3 Hxspiperone Ž0.5–30 nM.. The K d

K. Moroi et al.r Brain Research 794 (1998) 291–298

and Bmax values were obtained by linear regression analysis of the Scatchard data.

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8.0. followed by centrifugation divided vesicles into decoated vesicles Žcore. and the coat structure containing clathrins and AP2 proteins ŽFig. 1A..

2.4. SDS-PAGE and immunoblotting The G proteins present in coated vesicles were detected by immunoblotting with antibodies against the a-subunits of G proteins as previously described w27x with minor modifications. The coated vesicles were suspended in sample buffer containing 4% SDS, electrophoresed on 10% SDS-polyacrylamide gels, and transferred onto PVDF membranes. Strips of the membranes containing 5–10 mg of protein were incubated with antibodies against G proteins at 1:2000 dilution for 2 h at room temperature. After washing, the strips were incubated with horseradish peroxidase-conjugated secondary antibody before development by chemiluminescence. Protein in the gels was stained with Coomassie brilliant blue ŽCBB.. 2.5. Determination of adenylate cyclase actiÕity Adenylate cyclase activity was assayed according to the method described previously w29x. The coated vesicles Ž20–30 mg protein. were preincubated at 308C for 5 min in 100 ml of a reaction mixture consisting of 50 mM Tris– HCl, pH 7.4, 2 mM MgCl 2 , 1 mg mly1 bovine serum albumin, 1 mg mly1 of creatine kinase, 10 mM creatine phosphate, 0.1 mM Ro-20-1724, and 5 mM GppŽNH.p, 10 mM forskolin, and 5-HT or 8-OH-DPAT at various concentrations. The reaction was started by adding 100 mM ATP to each tube followed by incubation at 308C for 60 min. The reaction was stopped by boiling for 3 min, and the reaction mixtures were centrifuged at 12,000 = g for 10 min. Aliquots Ž25–50 ml. of supernatant were used to determine cyclic AMP level by radioimmunoassay. The background value was defined as the amount of cyclic AMP without the addition of ATP. All samples were run in duplicate.

3. Results 3.1. Characterization of coated Õesicles isolated from boÕine brain cortex The contents and purity of the coated vesicle preparations were examined by electron microscopy and SDSPAGE. Coated vesicles with a diameter of 70–90 nm accounted for approximately 98% of all the membranous components in the vesicle preparation wsee Ref. w29xx. SDS-PAGE was used to determine the major proteins of coated vesicles; the clathrin heavy and light chains, and the assembly polypeptides Žaccessory proteins, AP2.. Treatment of coated vesicles with 1 M Tris–HCl buffer ŽpH

3.2. [3H]5-HT and [3H]spiperone binding to coated Õesicles Binding of w3 Hx5-HT and w3 Hxspiperone to the coated vesicles reached equilibrium levels by 15–30 min at 308C. The binding was reversed by the addition of 100 mM 5-HT for w3 Hx5-HT or 10 mM ketanserin for w3 Hxspiperone. The binding of the radiolabeled ligands increased with increasing concentration of coated vesicles ranging from 100 to 500 mg of protein. Coated vesicles, 200–300 mg in protein content, were incubated at 308C for 30 min. Tritiated 5-HT and w3 Hxspiperone bound to the coated vesicles with high affinity and in a saturable manner ŽFig. 2A.. Scatchard analysis of binding data indicated that both ligands bound to a single class of sites. The apparent K d s were 2.4 " 0.3 nM for w3 Hx5-HT and 1.75 " 0.2 nM for w3 Hxspiperone ŽFig. 2B.. The Bmax values were 35.7 " 4.8 fmol mgy1 protein for w3 Hx5-HT and 65.9 " 7.1 fmol mgy1 protein for w3 Hxspiperone binding, respectively. The w3 Hx5-HT and w3 Hxspiperone binding sites in the coated vesicles were characterized with several agonists and antagonists acting on 5-HT receptors. The binding of w3 Hx5-HT was displaced by the reagents in a rank order of potency of 5-HT ) 8OH-DPAT) methysergide) mianserin but not by ketanserin ŽFig. 3A.. These findings were similar to those previously reported for 5-HT1A receptors in mammalian brain membranes w17,33x, and suggest the presence of 5-HT1 receptors, most probably 5-HT1A receptors, in the coated vesicles. The specific 5-HT1A receptor agonist, w3 Hx8-OH-DPAT, bound to the coated vesicles at 3.5 fmol mgy1 protein under the standard assay conditions Ž1 nM w3 Hx8-OH-DPAT., which corresponded to about 20% of the total w3 Hx5-HT binding Ž18.2 fmol mgy1 protein at 4 nM w3 Hx5-HT.. The w3 Hxspiperone binding was displaced by the reagents in a rank order of potency of spiperone) ketanserin) mianserin, but not by either 5-HT or 8-OH-DPAT even at 1 mM ŽFig. 3B.. A similar rank order of potency of the reagents for the 5-HT2A receptors was described in a review by Hoyer et al. w17x. In addition, similar findings were reported in mammalian cells transfected with a cloned rat 5-HT2A receptor cDNA w34x, suggesting that the coated vesicles contain 5-HT2A receptors. 3.3. Distribution of [3H]ligand binding in coated Õesicles After removal of the clathrin coat from the vesicles, ligand binding to the decoated vesicles and the coated structures was determined under the standard assay conditions described in Section 2. The binding activity observed

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Fig. 1. ŽA. Coomassie brilliant blue ŽCBB. staining of bovine brain coated vesicles after SDS-PAGE. CV; coated vesicles, core; decoated vesicles, coat; coat fraction. ŽB. Western blotting of a-subunits of G proteins by anti-G protein antibodies in coated versicles. lane 1; Ga s, lane 2; Ga il, lane 3; Ga i2, lane 4; Ga i3, lane 5; Ga o, lane 6; Ga qr11. Molecular masses of the marker proteins are shown on the left and right.

in the coated vesicles was recovered to the decoated vesicles and produced increases to 190% of w3 Hx5-HT and 170% of w3 Hxspiperone specific binding per mg protein as shown in Fig. 4. The elevation of the specific binding may be explained by the fact that the protein content of the

decoated vesicles constitutes about 60% of the total protein of the coated vesicles. These results indicated the presence of 5-HT1A and 5-HT2A receptors in the inner core vesicles originally derived either from the plasmalemma or the membranes of the Golgi apparatus.

Fig. 2. Saturation curve ŽA. and a Scatchard plot ŽB. of w3 Hx5-HT and w3 Hxspiperone binding to bovine brain coated vesicles. The binding experiment was performed as described in Section 2 over a concentration range from 0.5 to 30 nM. The points are representative of a typical experiment repeated five to six times in duplicate.

K. Moroi et al.r Brain Research 794 (1998) 291–298

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Fig. 3. Displacement of w3 Hx5-HT ŽA. and w3 Hxspiperone ŽB. binding to bovine brain coated vesicles by several agonists and antagonists. 5-HT ŽI., 8-OH-DPAT ŽB., spiperone Ž`., methysergide Že., mianserin Ž\., ketanserin Žv .. The binding experiment was performed as described in Section 2. Data are mean values from duplicate assays performed in a single experiment. Each experiment was repeated three to six times.

3.4. Detection of a-subunits of G proteins in coated Õesicles Serotonin receptors belong to the large family of G protein-coupled seven-transmembrane receptors, and their signals are transduced to the effector systems through G proteins. The 5-HT1 receptors, 5-HT1A , 5-HT1B and 5HT1D , are negatively coupled to adenylate cyclase via Giro-proteins w15,38–40x. The 5-HT1B receptor has been reported to be present in mice and rats but not in bovine

Fig. 4. Distribution of w3 Hx5-HT and w3 Hxspiperone binding to bovine brain coated vesicles ŽCV., decoated vesicles Žcore. and coat fraction Žcoat.. The core and coat were obtained after treatment with 1 M Tris–HCl buffer, and incubated with 4 nM w3 Hx5-HT or 1 nM w3 Hxspiperone as described in Section 2. Data are mean values from two experiments.

brain w16,17x. The 5-HT2A receptors are linked to phosphatidylinositol turnover via Gqr11-protein and phospholipase C w10,18x. w3 HxAgonist binding to 5-HT receptors is regulated by GTP, and the affinity of agonists to the 5-HT receptors decreases in the presence of GTP or GTP analogs w2,32x. It has also been reported that N-ethylmaleimide ŽNEM. inhibits w3 Hx5-HT binding to the 5-HT1A , 5-HT1B and 5-HT1D sites, but not to 5-HT1C sites or w3 Hxketanserin binding to the 5-HT2A receptors, by inactivating the G proteins associated with the 5-HT receptor subtype w43x. We examined whether G proteins were present in the coated vesicles and whether the 5-HT receptors present in the coated vesicles were coupled to adenylate cyclase activity. After SDS-PAGE, the coated vesicles were transferred onto PVDF membranes and G proteins were determined by western blotting using specific anti-G protein antibodies. As shown in Fig. 1B, the coated vesicles contained a-subunits of Gs, Gi2, Gi3, Go and Gqr11 protein. In the coat fraction, no bands were stained by these antibodies Ždata not shown.. w3 Hx5-HT binding to the coated vesicles was inhibited to 70% of the control in the presence of 100 mM GTP and was concentration-dependently inhibited by NEM from 10y7 to 10y4 M. These findings suggest an association of w3 Hx5-HT binding sites with G proteins in the coated vesicles. On the other hand, 100 mM GTP affected neither the amount of w3 Hxspiperone binding nor the affinity of displacement by 5-HT of w3 Hxspiperone binding to the coated vesicles. 3.5. Adenylate cyclase actiÕity in coated Õesicles Adenylate cyclase activity was detected in the coated vesicles with the basal activity of cyclic-AMP production

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Fig. 5. Effects of 5-HT and 8-OH-DPAT on forskolin-stimulated adenylate cyclase activity in bovine brain coated vesicles. Adenylate cyclase activity was measured as described in Section 2. Control activity in the presence of 10 mM forskolin was 0.20"0.03 pmol cyclic AMP productionrmg proteinrmin.

of 0.20 " 0.03 pmol mgy1 miny1 . The activity in the coated vesicles was less than 1% of those observed in rat and calf brain membranes w15,38–40x. 5-HT and 8-OHDPAT inhibited forskolin-stimulated adenylate cyclase activity concentration-dependently, and maximal inhibition amounted to about 20% ŽFig. 5., indicating that the w3 Hx5HT binding sites in coated vesicles are negatively coupled to adenylate cyclase activity.

4. Discussion Here, we presented evidence for the existence of 5-HT1A and 5-HT2A receptors in coated vesicles. In addition, we clarified the coupling of the 5-HT1A receptors to an adenylate cyclase activity via G proteins. The K d value of 2.5 nM for w3 Hx5-HT binding to the 5-HT1 receptors was the same as that Ž2.7 nM. reported in membrane preparations from the bovine frontal cortex w12x. The K d value of 1.75 nM for w3 Hxspiperone binding to 5-HT2A receptors was slightly higher than those Ž0.4–0.7 nM. reported in brain membranes w5,6,31x. On the other hand, the density of w3 Hx5-HT and w3 Hxspiperone binding sites, representing the 5-HT1 and 5-HT2A receptors, in the coated vesicles were about 10% of those reported in frontal cortex membranes of rat w6,33x, bovine w12,16x and human w5x brains. Similar findings showing small populations of neurotransmitter receptors in the bovine brain coated vesicles were observed for adenosine A 1 w13x and D1rD 2 w29x receptors. This low density may be in part due to a high turnover rate of coated vesicles in brain tissue.

The data from the displacement experiments of w3 Hx5HT and w3 Hxspiperone binding to the coated vesicles indicated the presence of 5-HT1A and 5-HT2A receptors. The bovine brain has, however, been reported to contain 5-HT1D sites which occupied 60–70% of the total 5-HT1 binding sites with high affinity to methysergide and mianserin w16,44x. w3 Hx8-OH-DPAT, a specific ligand of 5-HT1A receptors, bound to 20% of the total w3 Hx5-HT binding sites in the coated vesicles. Methysergide and mianserin suppressed w3 Hx5-HT binding as shown in Fig. 3A. Thus, we cannot exclude the 5-HT1D sites from the w3 Hx5-HT binding sites observed in bovine brain coated vesicles. w3 HxSpiperone labeled both 5-HT2A and dopamine D 2 receptors. It has, however, been demonstrated that in the rat frontal cortex and the superficial layer of the neocortex, w3 Hxspiperone labeled 5-HT2A receptors but not dopamine D 2 receptors w1,20x. Although dopamine D 2 receptor mRNA expressed in the rat neocortex at a low level, no D 2 receptors labeled by w3 Hxraclopride were observed in these areas w24x. In the human brain, 5-HT2A receptors were present in layers I to IV of the cortex at high density w5x. Taking these observations into consideration, the coated vesicles in the present study were prepared from the superficial layer of the neocortex and ketancerin, a 5-HT2A specific antagonist, was used to define w3 Hxspiperone binding to 5-HT2A receptors of the coated vesicles. w3 HxSpiperone binding to the coated vesicles may mostly represent binding to 5-HT2A receptors. The 5-HT1 and 5-HT2A receptors are members of a large family of plasma membrane receptors which are coupled to G proteins; 5-HT1A w15x and 5-HT1D w38x receptors are negatively coupled with an adenylate cyclase via Giro-proteins. 5-HT2A receptors are linked to phosphatidylinositol turnover via Gqr11-protein and phospholipase C w10,18x. In this study, we showed the presence of the a-subunits of G proteins, Ga i2, Ga i3, Ga o and Ga qr11, and of adenylate cyclase activity in the coated vesicles. The activities stimulated by forskolin were dosedependently inhibited by 5-HT and 8-OH-DPAT. The Giro proteins in the coated vesicles are present in a functional arbrg complex form which is ADP-ribosylated by pertussis toxin w27x. w3 Hx5-HT binding to the coated vesicles was reduced with the addition of 100 mM GTP and 100 mM NEM, indicating that the binding sites were associated with G proteins w2,32,43x. Together with these findings, it is concluded that the 5-HT1A receptors in the coated vesicles are negatively coupled to an adenylate cyclase activity through Giro-proteins. However, the association of w3 Hxspiperone binding sites with G proteins was not clear, because 100 mM GTP affected neither the amount of w3 Hxspiperone binding nor the affinity of displacement by 5-HT of w3 Hxspiperone binding to the coated vesicles. Agonist-induced desensitization is a common feature of G protein-coupled signal transduction systems. Desensitization of b 2-adrenergic receptors has been extensively

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studied w22x. After exposure to agonists, b 2-adrenergic receptors are phosphorylated by cyclic AMP-dependent protein kinase, protein kinase C or G protein-coupled receptor kinases, resulting in dissociation of the effectors from the receptors. The phosphorylated receptors consequently bind to cytosolic proteins referred to as b-arrestins, followed by internalization via dynamine-associated coated vesicle formation w45x. Recently, b 2-adrenergic receptors have been demonstrated to bind directly to clathrins through b-arrestins instead of AP2 w14x, which plays an essential role in endocytosis. This strongly suggests that other seven-transmembrane receptors coupled with G proteins may be internalized via clathrin-coated vesicles after agonist-induced phosphorylation. Repeated or prolonged exposure of 5-HT receptors to agonists decreases their responsiveness Ždesensitization.. Recently, Raymond w36x and Nebgil et al. w28x reported that exposure of 5-HT1A receptors to agonists resulted in rapid phosphorylation of the receptors by both protein kinase C and G protein-coupled receptor kinases. Rahman and Neuman w35x demonstrated that desensitization of 5HT2A receptors was caused by activation of protein kinase C and that internalization of those receptors after 5-HT exposure was involved in the mechanism of desensitization. Berry et al. w4x demonstrated the agonist-induced internalization of 5-HT2A receptors via the endosome pathway by histochemical techniques using an anti-5-HT2A receptor antibody. Single or repeated administration of mianserin, a 5-HT2A receptor antagonist, has been reported to cause a decrease in the density of 5-HT2A receptors Ždown-regulation. w6x, and at the same time a reduction in phosphoinositide hydrolysis response to 5-HT mediated by 5-HT2A receptors in rat brain w9x. At present, however, it has not been confirmed whether internalization of the receptors mediated by the coated vesicles is involved in the down-regulation of G protein-coupled receptors after exposure to antagonists w22x. Our results demonstrated the presence of 5-HT1A and 5-HT2A receptors in coated vesicles prepared from the bovine brain. Our findings suggested that these receptors are endocytosed via coated pit formation possibly in a manner similar to that reported for b 2-receptors w11,14x, that 5-HT1A receptors are internalized in association with G proteins and adenylate cyclase activity, and that the coated vesicles are presumably involved in the mechanisms of desensitization or down-regulation of 5-HT1A and 5-HT2A receptors.

Acknowledgements We thank Dr. S. Kimura for his support of this project. This work was supported in part by the Uehara Foundation and by the Pharmacopsychiatry Research Foundation ŽNihon Zohki, Osaka..

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