Molecular pharmacology of AMPA receptor trafficking proteins - TARPs - evidence for an association with 5HT(_2c) receptors

Abstract

There has been an increasing awareness of the involvement of neurotransmitters other than serotonin in depression, with new antidepressants possessing effects on other receptor types, such as Agomelatine®, an antagonist of 5HT(_2c) receptors that functions as an agonist at melatonin receptors. AMPA receptors are one of the families of ionotropic glutamate receptors and another neurotransmitter receptor type that have been demonstrated to be important in the function of new antidepressants. AMPA receptors possess effects upon brain-derived neurotrophic factor (BDNF) expression, a protein involved in neurogenesis in the hippocampus, which is believed to be pivotal to antidepressant efficacy, with BDNF expression diminishing in critical brain areas in response to chronic stress, but increasing in the hippocampus in response to treatment with both antidepressants and/or AMPA receptor modulators. AMPA receptors interact with a family of accessory proteins, the transmembrane AMPA receptor regulatory proteins (TARPs), which not only possess important roles in the trafficking and targeting of AMPA receptors, but also function as auxiliary subunits to AMPA receptors. Present in several isoforms, each individual TARP also directly modifies AMPA receptor kinetics. As such TARPs and their effects must be taken into account for any pathophysiological or drug-induced change involving AMPA receptors. Despite the vast literature on AMPA receptors, there is comparatively little information regarding how TARPs modify AMPA receptor function, largely due to the absence of the necessary tools. We developed polyclonal antibodies specific to each of the known TARP isoforms (y2, y4, y8) mapping the distribution of the TARPs in the mouse CNS, displaying a different distribution profile for each of the TARP isoforms. TARP y8 is of particular interest, being shown to have a wide expression in the CNS from the frontal cortex to the spinal cord, but also a regional distribution in the forebrain that shares similarities to a positive allosteric modulator of AMPA receptors. There is also some evidence of a strain dependent distribution of TARP 78, possibly contributing to some of the behavioural differences between strains. With the extensive distribution of TARP y8 in the forebrain, particularly in those structures shown to experience severe neuronal atrophy in depression, such as the hippocampus, we focused on the antibodies generated to this isoform to generate immunoaffinity columns and immunopurify TARP y8 and its interacting proteins from Triton X-100’_™’ solubilised, so effectively non-synaptic, cerebral cortex. Examination of the purified TARP y8 and its interacting partners by both immunological and proteomic techniques revealed a range of proteins previously not implicated as TARP interacting proteins important in several pathophysiological situations, including several isoforms of actin. Furthermore, the immunopurified TARP y8 material also contained a protein identified with multiple 5HT(_2c) receptor antibodies at -60 kDa, the molecular weight correlating to fully glycosylated 5HT(_2c) receptor. Further study of TARP and AMPA receptor levels in the forebrain of mice with either forebrain-specific over-expression, or forebrain-specific knockdown of 5HT(_2c) receptors, identified several differences in total protein levels of the TARPs and AMPA receptor subunits. TARP y8 was shown to possess higher levels of expression in both of the mice strains with altered 5HT2c receptor expression, suggesting a complex functional interaction between TARPs/AMPA receptors and 5HT(_2c) receptors. These results, in addition to providing evidence of strain variations with regard to TARP distributions, have also identified several previously unknown TARP y8 interacting proteins, including, but not limited to cytoskeletal proteins. The results also show evidence of both a physical and functional interaction of TARP y8 and AMPA receptors with 5HT(_2c) receptors in the forebrain, particularly the cerebral cortex - findings of potential importance regarding the role of AMPA receptors in mood disorders.