Involvement of the Third Intracellular Loop of 5HT2C Receptor in the Physical Interaction Between 5HT2C Receptor and PTEN

To test the hypothesis, the first question we have to answer is whether PTEN interacts physically with 5-HT2C receptors. Because PC12 cells express both PTEN (Lachyankar et al. 2000; Musatov et al. 2004) and 5-HT2C receptors

(Flomen et al. 2004), we tried to identify the possible existence of PTEN:5-HT2C receptor complexes in cultured PC12 cells using coimmunoprecipitation (Co-lP) assay. A complex was immunoprecipitated from PC12 cell lysates by a 5-HT2C receptor antibody, and the existence of PTEN in the complex was confirmed via Western blotting using a PTEN antibody (Ji et al. 2006). In parallel, 5-HT2C receptor was immunoprecipitated from the PC12 cell lysates by the PTEN antibody (Ji et al. 2006). These results demonstrate a physical interaction between PTEN and 5-HT2C receptors.

Then, we tried to determine the exact domain of 5-HT2C receptor responsible for its interaction with PTEN employing a pull-down assay, which utilizes a purified and tagged or labeled bait protein to generate a specific affinity support able to bind and purify a prey protein from a lysate sample, or other protein-containing mixtures. In an attempt to determine the site of interaction, a fusion protein incorporating the carboxyl-terminal tail of the 5-HT2cR was synthesized. The belief that this particular section was responsible for the interaction between PTEN and 5-HT2C receptor was due to the knowledge that the C-terminal of GPCRs commonly contains protein residues that couple with other proteins (Lee et al. 2002, 2003). Unfortunately, our original pull-down assay excluded a direct binding of PTEN with the C-terminal domain of 5-HT2C receptors. We then focused on the third intracellular loop of the 5-HT2C receptor, which is prominently longer than the C-terminal region. A pull-down assay utilizing two fusion proteins (one containing the full sequence of the C-terminal, the other comprising the entire sequence of the third intracellular loop) showed that the third intracellular loop, but not the C-terminal or GST alone, precipitated PTEN from PC12 lysates (Ji et al. 2006), thereby suggesting the critical involvement of the third intracellular loop of 5-HT2C receptor in the physical interaction between 5-HT2C receptor and PTEN.

While a motif of several amino acids is usually required for protein-protein interaction, the third intracellular loop of 5-HT2C receptor consists of 87 amino acids. We therefore tried to identify the exact motif within the third intracellular loop of 5-HT2C receptor that is responsible for the physical interaction between 5-HT2cR and PTEN. The third intracellular loop was split into five segments: 3L1F (Leu237-Gly252), 3L2F (His253-Asn267), 3L3F (Cys268-Asn282), 3L4F (Pro283-Arg297), and 3L5F (Pro298-Lys313). Each segment was integrated into a fusion protein and a pull-down assay using PC12 cell cultures demonstrated that 3L4F, but not 3L1F, 3L2F, 3L3F, 3L5F, or GST alone, precipitated PTEN (Ji et al. 2006). The above experiments provide the first evidence that 5-HT2C receptor physically interact with PTEN through the third intracellular loop of 5-HT2C receptor, but these experiments did not answer the question whether 5-HT2C receptor interact with PTEN directly or indirectly though additional protein(s). To answer this question, we conducted an in vitro pull-down assay in which only purified PTEN and testing fusion proteins were added, so that if PTEN is precipitated by one or more fusion proteins originating from 5-HT2C receptor, it is strongly suggestive that PTEN directly interact with 5-HT2C receptor. We showed that PTEN was selectively precipitated by the fusion proteins containing the third loop and

3L4F, suggesting the necessity of the 3L4F motif in mediating the direct interaction between PTEN and 5-HT2C receptor (Ji et al. 2006). All the data collected so far just indicate the importance of 3L4F in mediating 5-HT2C receptor coupling PTEN, but it is still unknown whether 3L4F is the only motif in 5-HT2C receptor that makes major contributions to protein-protein interaction between PTEN and 5-HT2C receptor. This is because we have not examined whether other domains of 5-HT2C receptor, including the first or second intracellular loop, transmembrane domains, extracellular loops, or extracellular N-terminal, may also contain motif(s) responsible for 5-HT2C receptor coupling with PTEN. Our further experiment demonstrated that addition of synthesized 3L4F to PC12 cell culture lysate before the conduction of Co-IP completely blocked Co-IP between 5-HT2C receptor and PTEN (Ji et al. 2006). Not only do these data provide convincing evidence indicating the importance of 3L4F in the interaction of 5-HT2CR with PTEN, but they also suggest that 3L4F can be used to competitively disrupt the interaction between PTEN and 5-HT2cR.

In summary, we have established using PC12 cells that 5-HT2C receptor interacts directly with PTEN through the 3L4F motif of its third intracellular loop. These results led us to further explore whether 5-HT2cR also forms heterodimer with PTEN through 3L4F in the rat VTA, a brain region critically involved in drug reward. We found that similar to PC12 cell culture, addition of synthesized 3L4F to VTA lysate before the conduction of Co-IP completely blocked Co-IP between 5-HT2C receptor and PTEN (Ji et al. 2006), suggesting that 5-HT2C receptor:PTEN heterodimers exist in the rat VTA and could be disrupted by the 3L4F peptide.

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