Abstract||The serotonin receptor 2A (5-HT₂[A]) is a member of the G-protein coupled receptor family and is of interest due to its role in physiological functions such as smooth muscle contraction, platelet aggregation, thermoregulation, learning and memory. More importantly, 5-HT₂[A] has also been implicated in CNS disorders including schizophrenia, depression and anxiety.
A yeast two-hybrid screen had previously been carried out to identify proteins that interacted with 5-HT₂[A] and therefore may modulate intracellular function. The cytoskeletal actin-binding protein filamin-C was identified as a possible 5-HT₂[A] interacting partner. The aim of the research in this thesis was to further investigate the potential interaction between 5-HT₂[A] and filamin-C and to investigate functional roles for the interaction.
A fragment of human filamin-C, aa 2162-2725, was shown to interact with the C-terminus of human 5-HT₂[A] using two in vitro techniques, the yeast-two hybrid system and a GST capture assay. The region of filamin-C needed to bind to 5-HT₂[A] was narrowed to the start of repeat 20, aa 2251, through to aa 2424 at the beginning of repeat 22 and comprises 182 residues. The 5-HT₂[A] region needed to bind to filamin-C was ascertained via yeast two-hybrid to be 31 amino acids between 394-423.
Work was performed to determine whether FLNC mRNA was expressed in neural and glial cells and whether FLNC and HTR2A mRNA were co-expressed in any cells. FLNC mRNA was identified in seven out of eight neural and glial cell lines and western blot analysis confirmed this finding at the protein level. Two cell lines, U-118MG and A172, were found to contain both HTR2A and FLNC mRNA. Co-immunoprecipitation experiments showed endogenous filamin-C bound to endogenous 5-HT₂[A] and this complex could be precipitated using anti-filamin-C antibody. Additionally, a GST-5-HT₂[A] fusion complex was found to bind to endogenous filamin-C from U-118MG cells.
Immunofluorescent labelling of cells was used to study filamin-C and 5-HT₂[A] proteins in vivo. U-118MG cells showed staining for 5-HT₂[A] around the membrane of the cell, as well as in the cytoplasm, whereas filamin-C staining occurred in the cytoplasm. Co-localisation analysis identified some areas of overlap between 5-HT₂[A] and filamin-C in the cytoplasm of U-118MG cells.
The functional role for the 5-HT₂[A]/filamin-C colocalisation was investigated. It was postulated that filamin-C may be involved in the internalisation of 5-HT₂[A]. To test this hypothesis, an in vivo model system was used to investigate whether disruption of the filamin-C/5-HT₂[A] interaction affects internalisation of the receptor. The key preliminary findings of this study, which used expression of a competitor peptide, to disrupt and co-interact, suggested that the filamin-C/5-HT₂[A] interaction is not essential for the internalisation of receptors in response to ligand binding. However, this interaction was important for delivery or maintenance of 5-HT₂[A] to the cell membrane, and expression of the competing peptide caused an accumulation of cytoplasmic 5-HT₂[A].