Internalisation of the type II gonadotropin-releasing hormone receptor of marmoset monkey

Doctoral Thesis


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University of Cape Town

The mammalian type II GnRH receptor has a C-terminal tail unlike the mammalian type I GnRH receptor, which uniquely lacks the cytoptasmic C- terminal domain. lnternalisation of a mammalian type ll GnRH receptor has never been investigated, therefore this thesis studies the internalisation pathway of the type ll GnRH receptor. As the C-terminal tail mediates rapid internalisation of many G protein-coupled receptors this research investigates the functional role of the C-terminal tail and intracellular loop in receptor internalisation. The internalisation pathway of the type ll GnRH receptor in COS-1 cells was investigated by co expressing dominant negative mutants and wild- type constructs of G protein-coupled receptor kinases (GRKs), dynamin-1 and β-arrestin 1 and 2 with the type II GnRH receptor. The results show that internatisation of the receptor requires GRK 2 and dynamin but does not require β-arrestin 1 and 2. Furthermore, inhibitors to both the caveolae pathway as well as the clathrin coated vesicle endocytosis abolished receptor internalisation indicating that both structures are involved in internalisation of the receptor. Even though in COS-1 cells the type ll GnRH receptor internatises in a β-arrestin independent manner, internalisation of this receptor can be enhanced by over-expression of wild type β-arrestin. This indicates that the type ll GnRH receptor is able to utilise a β-arrestin mediated internaltsation pathway if high levels of β-arrestin are present in the cell. The mammalian type ll GnRH receptor internalises with enhanced rate and extent compared to the tail-less human type I GHRH receptor. The role of the C-terminal tail of the type ll GnRH receptor in internalisation was investigated by measuring internalisation of C-terminally truncated mutants. It was found that the region between Gly 343 and Ser 335 within the C-terminal domain is important for receptor internalisation. Substitution of putative phosphorylation sites within this region revealed that Ser 338 and Ser 339 are critical for rapid receptor internalisation. Furthermore a serine residue in intracellular loop three (Ser 251) was shown to play a role in signalling as well as in internalisation. Since dominant negative GRK 2 could not inhibit internalisation of a mutant lacking all three serine residues, but could reduce internalisation of the wild-type receptor, we suggest that Ser 251, 338 and 339 are target of phosphorylation by GRK. However these phosphorylation sites as well as the C-terminal tail are not necessary for β-arrestin dependent internalisation. Taken together this thesis elucidates the internalisation pathway of a mammalian type lI GnRH receptor and identified residues within the C-terminal tail and intracellular loop three that are critical for rapid internalisation.

Bibliography: leaves 102-124.