Complement receptor type 2 (CR2 CD21) is a type 1 membrane-bound glycoprotein in the regulators of complement activity (RCA) family. One of its ligands is C3d, a physiological cleavage fragment of the central complement protein C3. The extracellular portion is comprised of 15 or 16 short complement repeat (SCR) domains. Six of the 14 linkers between these SCR domains are atypically long and the implications of this on the solution structure and function of CR2 was unknown. Two previous crystal structures of CR2 SCR 1-2 showed the two SCR domains formed contacts with each other in a closed V-shape. In this thesis, three different recombinant constructs of CR2 were studied in solution using X-ray and neutron scattering, analytical ultracentrifugation, and constrained modelling. The combination of these techniques resulted in medium resolution structures for each of CR2 SCR 1-2, CR2 SCR 1-2 in complex with C3d, a CR2-Ig chimaera, and CR2 SCR 1-15. It was determined that the longest linkers within CR2 are highly flexible. CR2 SCR 1-2 was shown to exist in an open-V shape conformation, both free and in its complex with C3d. Studies on free C3d in solution showed that this forms concentration dependent dimers, although this dimerisation does not occur in the CR2 complex. The modelled complex between C3d and CR2 SCR 1-2 showed that SCR1 as well as SCR 2 is capable of interacting with the ligand. The chimaera of fully glycosylated human CR2 SCR 1-2 with the mouse IgGl Fc domain confirmed the results for the solution structure of free CR2 SCR 1-2 and gives an insight into the therapeutic possibilities of such a molecule. Modelling of glycosylated full length CR2 revealed a flexible semi-extended arrangement of the 15 SCR domains. These results provide an explanation of the molecular basis for the interaction of CR2 SCR 15 with C3d and three other ligands, CD23, gp350 and IFN-ct, in terms of proximity relationships between neighbouring SCR domains. Insight was also obtained into the possible mechanisms by which CR2 SCR 1-15 may activate B cells.

y-amino-butyric acid type A (GABAA) receptors are the major sites of fast synaptic transmission in the central nervous system and can be assembled from 7 subunit classes: al-6, (31-3, yl-3, 5, e, n, and 6. Although expression of receptor a and (3 subunits can produce functional GABA-gated chloride channels the presence of the y2 subunit within the GABAA receptor complex has been shown to play a key role in clustering and synaptic targeting. The regulation of GABAA receptor cell surface stability by endocytosis has been previously shown to be a crucial determinant of inhibitory synaptic strength. The main focus of this thesis is to further understand the molecular mechanisms by which the number of GABAA receptors clustered in synapses can be regulated. Microscopy and biochemical experiments showed that the y2 subunit of GABAA receptors confers an enhancement in the targeting of GABAA receptors to a degradative pathway after internalization from the plasma membrane. Furthermore, blocking lysosomal degradation with the lysosomal inhibitor leupeptin results in increased GABAergic currents in cells expressing GABAA receptors containing ocl, (33 and y2 subunits but not those expressing a 1(33 receptors alone. In order to characterise the molecular signals determining this degradative pathway, (33-y2 chimeras were created and expressed in both HEK293 cells and hippocampal neurons. Quantitative confocal microscoscopy studies on these chimeras revealed a 20 amino acid region to be responsible for the late endosomal/lysosomal targeting of y2 subunit containing GABAA receptors. Further analysis into this 20 amino acid region identified a lysine stretch that when mutated resulted in decreased levels of internalised GABAA receptors in late endosomes. Within this region the mutation of serine 327, a known phosphorylation site of GABAA receptors, also caused a reduction in the targeting of internalised GABAA receptors to lysosomes. Interestingly GABAergic currents formed by the expression of a 1(33 and y2L (lysine or serine mutant) were unaffected by the lysosomal inhibitor leupeptin. Biochemical studies found these lysines to be modified by the small poly-peptide ubiquitin, a known molecular signal for the degradation of proteins in the proteasome and the endocytic pathway. Moreover, mutation of serine 327 was sufficient to inhibit the ubiquitination of this lysine stretch in the intracellular loop of the y2L subunit. The results presented in this thesis suggest that the endocytic sorting fate of synaptic GABAA receptors plays an important role in determining the strength of inhibitory synapses and provide a mechanism by which serine 327 acts as a molecular switch to regulate the ubiquitination dependent degradation of GABAA receptors in the lysosome.

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