Principio de Proporcionalidad en Sentido Estricto

or aggregates as shown in figure 39. It is interesting to note that the NMDAR anchoring protein a-actinin-2 forms dimers in a similar way as shown in fig 36C). Although there is no direct evidence that unprocessed M AP IB exists in vivo, it seems likely that if it is synthesised in the form of a polyprotein, then a small proportion of it will rem ain in this form. Given the availability of an anti-LC l antibody, an interesting experiment to test this hypothesis would be to carry out a simple pull-down assay with G S T -pl from retinal extract, and probe the western blot with anti-LC l as well as anti-M A PlB . This would indicate whether or not LC l is still covalently attached to the HC C-terminus, rather than non-covalently associated with the N-terminus after proteolytic processing.

Comparison with other neurotransmitter receptor clustering mechanisms.

An attempt was m ade in chapter 1 to com pare some of the characteristics o f neurotransm itter receptor clustering mechanisms. The m olecular structures of clustering proteins seem to be very diverse, and M A PIB does not share any sequence hom ology with any o f the other known proteins. It is clear that interacting with GABA^Rs is not the only function of M A PIB ; it has a very im portant developm ental role in the process o f neuritogenesis (Gordon-W eeks, 1997; Ulloa et al., 1997). Gephyrin must also have other functions; it is found at focal adhesions in spinal cord neurons at early developm ental stages and is expressed in a wide range of tissues which do not express GlyRs (Kirsch et al., 1993a; Prior et al., 1992). In hippocam pal neurons, a-actinin-2 is found not only associated with NM DARs at the PSD, but also with m icrotubules in dendritic shafts (Wyszynski et al., 1998). This poses the same question as in the p l/M A P lB situation; how do receptors avoid an interaction with the anchoring protein along the length o f the neurite, but interact at the synapse? The N M D A R /a-actinin-2 interaction is disrupted by Ca^Vcalmodulin, but this is more likely to occur at the synapse than in the dendritic shaft, suggesting that there may be some other regulatory mechanism. M A PIB has the potential for an interaction with both actin and tubulin (Fujii et al., 1993; Pedrotti et al., 1996), which has also been implied for gephyrin (Kirsch and Betz, 1995), and the observation that a-actinin-2 decorates m icrotubules in dendritic spines suggests that it can also bind both cytoskeletal elements (W yszynski et al., 1998). It is interesting that the behaviour o f M A P lB /p l complexes in fibroblasts is similar to that o f gephyrin/GlyR, rapsyn/neuronal nAchR and SAP97/NMDAR; that is, it forms intracellular aggregates rather than surface clusters (Feng et al., 1998; Kim and Sheng, 1996; Kirsch et al., 1995). This could reflect a

similarity in the clustering process or in the mode of interaction with the cytoskeleton fo r these synaptic complexes.

It is useful to compare well-established anchoring mechanisms because they m ight give clues about further properties o f the p i/M A P IB interaction. However, the distinct functions o f each receptor type would suggest that the differences in the way the interactions are maintained and regulated will be more interesting than the similarities. This is highlighted by the fact that com pletely unrelated proteins m ediate a similar process for each type o f neurotransm itter receptor.

GLYT-1 and “clone 14”.

In addition to M A PIB, the yeast two-hybrid screen isolated two further p i-in teractin g clones; clone 6, which is a C-term inal variant o f the glycine transporter GLYT-1 E/F, and clone 14, which is a portion of a novel protein. The p l-G L Y T -IE /F interaction has been confirmed by in vitro overlay assay, and GLYT-1 E/F has been localised to the plexiform layers o f the retina. The interaction awaits further in vivo analysis. It would be o f great interest to analyse the influence

pi

subunit has on GLYT-1 function, and vice versa. Unfortunately, the antibody raised against clone 6 is not o f high quality, which m ight hinder in vivo investigation. Clone 14 is potentially very interesting because it interacts with

pi

and

p2 subunits (but not GABA^R a l , pS or y2). There is still no strong evidence

indicating the subunit composition o f GABA^Rs in vivo', there is the possibility o f

pi, p2

and

p3

hom omeric receptors, or heteromers form ed from com binations o f these. W hichever is the case, clone 14 is likely to be a protein involved in the biology of all GABAcRs, rather than a GABA^R subtype. The identity o f the full-length clone for this novel protein, and potential functions with respect to GABA^R biology should be investigated.

This study has isolated three GABAcR-interacting proteins, M A PIB, GLYT-1 E/F, and “clone 14” . The majority of the work has involved the characterisation o f the M A PIB interaction, which represents the first cytoskeletal anchoring m echanism for a GABAR.

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