Hydrophobic modification of the 5-HT 7(a) receptor

To examine whether the 5-HT7(a) receptor is acyl-modified, Sf.9 cells infected with recombinant or wild type baculovirus were metabolically labelled with [³H]-palmitic acid.

The resulting fluorogram (Fig. 3.3, right panel) demonstrates that the 5-HT7(a) receptor effectively incorporated [³H]-palmitate and that the labelled polypeptide co-migrated with the [³⁵S]-methionine labelled 5-HT7(a) protein.

Figure 3. 3. Palmitoylation of the 5-HT_7(a) receptor. Sf.9 cells expressing the 5-HT_7(a) receptor were labelled with [³H]-palmitate or [³⁵S]-methionine. Receptor was immunoprecipitated with anti-Myc antibodies and subjected to SDS/PAGE and to fluorography. Cells infected with a wild-type baculovirus (Bac) were used as a control. The fluorogram is representative of four independent experiments.

To distinguish between amide-type and ester type fatty acid linkages the chemical nature of the fatty acid bond in the 5-HT7(a) receptor was determined. In contrast to the amide bond, S-ester and hydroxyester linkages are sensitive to the presence of 2-mercaptoethanol (Schmidt et al., 1979). To investigate whether the fatty acid was attached to the 5-HT7(a) receptorby a S-ester bond, proteins labelled with [³H]-palmitic acid were subjected to 2-mercaptoethanol treatment. The results shown in Figure 3.4 A demonstrate that [³H]- palmitate-derived radioactivity bound to the protein was sensitive to heating with buffer supplemented with increasing concentrations of 2-mercaptoethanol. This suggests 5-HT7(a) Bac 5-HT7(a) Bac

[³⁵S]-Met [³H]-Pal 61

49

36 kD

that the 5-HT7(a) receptor contains exclusively S-ester –linked acyl groups and no fatty acids linked by an amide bond, which would have been resistant to such treatment.

Figure 3. 4. Fatty acid analysis.

A. Sf.9 cells expressing the 5-HT_7(a) receptor were labelled with [³H]-palmitate. After immunoprecipitation, samples were treated with increasing concentration of β-mercaptoethanol for 30 min at 37°C followed by SDS/PAGE and fluorography. The data shown are representative of three experiments.

B. After labelling with [3H]-palmitate cells, expressing the 5-HT_7(a) receptor were lysed, immunoprecipitated and subjected to SDS/PAGE. Gel was treated with 1 M Tris/HCl (pH 7.4) or 1 M hydroxylamine (pH 7.4).

The resulting fluorogram is representative of three independent experiments.

C. Receptor-bound fatty acids were analysed by thin - layer chromatography (TLC). The fluogram obtained from the RP 18 TLC plate after two days exposure was analysed with Gel-Pro Analyser software, version 3.1.

The S-ester bond can be distinguish from the hydroxyester one by its sensitivity to treatment with hydroxylamine (Kaufman et al., 1984). After treatment of gels containing fatty-acid labelled 5-HT7(a) protein with neutral hydroxylamine, this reagent cleaved the palmitate-derived label from the receptor, whereas labelled lipids remained unaffected (Fig. 3.4 B). This sensitivity to neutral hydroxylamine and reducing agent indicates that fatty acid is bound to the 5-HT7(a) receptor via an S-ester-type linkage.

To prove the identity of the fatty acids bound to the 5-HT7(a) receptor after labelling with [³H]-palmitate, proteins were subjected to fatty acid analysis. Fatty acids were hydrolysed from gel-purified the 5-HT7(a) receptor and separated into the individual fatty

β-mercaptoethanol (%)

acids species by TLC. Radio-chromatogram scanning of the TLC plates revealed that the 5-HT7(a) receptor contained only palmitic acid (Fig. 3.4 C).

3.2.1. Palmitoylation of the 5-HT7(a) receptor is agonist-dependent

Palmitate can be attached to the proteins in constitutive or in the reversible manner.

To test whether palmitoylation of the 5-HT7(a) receptorisdynamic modification, receptor was labelled with [³H]-palmitate during 60 minutes in the absence or in the presence of increasing concentrations of agonist 5-HT. The intensity of incorporated radioactivity was valuated from fluorograms exposed on gels containing immunoprecipitated the 5-HT7(a)

receptor after labelling. The data obtained demonstrate that treatment with agonist results in dose-dependent increase in incorporation of [³H]- palmitate into receptor (Fig. 3.5). It should be also noted that such increase in labelling can not be explained due to increase in the amount of newly synthesized receptor. Indeed, as shown in the Figure 3.5. B, the incorporation of [³⁵S]- methionine into the 5-HT7(a) receptor was even decreased after the application of agonist.

To exclude the influence of possible changes in the protein synthesis, cells expressed receptor were labelled with [³H]-palmitate or [³⁵S]-methionine in the presence or in the absence of protein synthesis blocker cycloheximide. As it is seen in resulting fluorogram (Fig. 3.6), block of the protein synthesis by cycloheximide has no effect on the 5-HT7(a) receptor palmitoylation. More important, incorporation of [³H]-palmitate still remains agonist-promoted. This result demonstrates that palmitoylation of the 5-HT7(a)

control 10nM 100nM 1µM 10µM 100µM 1mM

[³H-Pal]

control 5-HT(10 mM)

[³⁵S]-Met

Figure 3. 5. Agonist-promoted incorporation of [³H]-palmitate into the 5-HT7(a).receptor.

A. Sf.9 cells expressed the 5-HT7(a) receptor were labelled with [³H]-palmitate for 60 min in the absence or in the presence of increasing concentrations of 5-HT. The receptor was immunoprecipitated, separated by SDS/PAGE and subjected to fluorography.

B. The effect of 5-HT treatment on receptor synthesis.

A

B

receptor is post-translational process and indicate that previously synthesized receptor is available for repeated rounds of palmitoylation/ depalmitoylation.

3.2.2. Dynamic of the 5-HT7(a) receptor palmitoylation

In order to obtain more information about the dynamic of receptor palmitoylation, Sf.9 cells expressed the 5-HT7(a) were incubated with [³H]-palmitic acid for 5, 20, 40 and 60 min. As shown in Fig. 3.7, intensity of radioactivity of immunoprecipitated receptor increases steadily as a function of time reflects a basal palmitate turnover. To determine whether the activation of receptor influenced its palmitoylation state, the [³H]- palmitate incorporation was analysed after agonist stimulation. Results of this experiment reveal that palmitoylation of the 5-HT7(a) receptor was significant increased in the presence of 5-HT as compared with non-stimulated receptor (Fig. 3.7).

Cycloheximide - - + + - - + + 5-HT - + - + - + - +

[³H]-Pal [³⁵S]-Met

Figure 3. 6. Palmitoylation of the 5-HT7(a) receptor is independent from the protein synthesis.

Sf.9 cells expressing the 5-HT7(a) receptor were labelled for 60 min with [³H]-palmitate or [³⁵S]-methionine in the absence (-) or in the presence (+) of cycloheximide (50 µg/ml). In parallel, 100 nM 5-HT or water was added. After SDS/PAGE, radiolabel incorporation was detected by fluorography. Experiment was repeated three times.

Time (min) 5 30 60 90 Control

5-HT

Figure 3. 7. Time course of the incorporation of [³H]-palmitate into the 5-HT7(a) receptor after agonist treatment. Insect cells expressing the 5-HT7(a) receptor were labelled with [³H]-palmitate in the presence of 100 nM 5-HT for the periods indicated. Data was obtained after fluorography followed by immunoprecipitation and SDS/PAGE and represents of three independent experiments

3.2.3. Identification of acylation site(s) on the 5-HT7(a) receptor palmitoylation

Taken together, these data suggest that palmitoylation of the 5-HT7(a) receptor is dynamic process and agonist stimulation increase the rate of receptor palmitate turnover.

Given that the 5-HT7(a) receptor contains covalently-bound palmitate, the next step was to identify sites of acylation. The 5-HT7(a) receptor possesses three cysteine residues (Cys⁴⁰⁴, Cys⁴³⁸ and Cys⁴⁴¹) within its intracellular C-terminal domain and these cysteines can represent palmitoylation sites. To identify which of these cysteines can be palmitoylated, a number of substitution mutants (Cys⁴⁰⁴-Ser, Cys^438-441 and Cys404-438-441), in which different cysteine residues were replaced by serines was constructed.

Figure 3. 8. C-terminal sequence of the 5-HT7(a) receptor with substituted cysteine residues. Schematic view of the 5-HT7(a) mutants. The cytoplasmic, carboxyl-terminal sequences of the 5-HT7(a) receptor and three substitution mutants are given in a single-letter code. The amino acid numbers for four cysteine residues are indicated.

All resulting mutants were expressed by Baculovirus expression system and proteins were detected by Western blot analysis (Fig. 3.9).

Figure 3. 9. Expression and acylation of wild type and mutants of the 5-HT7(a) receptorin Sf. 9 cells.

A. Insect cells expressed wild type (wt) and different C-terminal cysteines mutants of the 5-HT7(a) receptor were lysed, separated by SDS/PAGE and then subjected to Western-blot analysis.

B. Sf.9 cells expressed the 5-HT7(a) receptor wild type and cysteine mutants were labelled with [³ H]-palmitate. Proteins were immunoprecipitated with Myc-antibodies and subjected SDS/PAGE and subsequent fluorography. Data shown are representative of two independent experiments.

S

To determine the site(s) of palmitoylation, individual mutants were metabolically labelled either with [³H]-palmitate or with [³⁵S]-methionine. Labelling with [³⁵S]-methionine shows that all mutants were expressed at the levels comparable with those of the 5-HT7(a) receptor wild type. Resulting fluorogram after labelling with [³H]-palmitate demonstrated that replacement each of single cysteine and replacement of several cysteines in different combinations results in decreased palmitoylation. However, even after replacement of all three C-terminal cysteines by serine (Cys404-438-441-Ser mutant) receptor was still palmitoylated (Fig. 3.9). Therefore, it can be concluded that in addition to the C-terminal cysteine residues, the 5-HT7(a) receptor is palmitoylated on additional cysteine located somewhere else. Similarly results has been previously reported for Gαs subunit (Kleuss and Krause, 2003) and for vasopressin V1a receptor (Hawtin et al., 2001).