Blassberg, R.A.; (2010) The mechanism of signal transduction by the GPI anchored Prod 1 protein. Doctoral thesis , UCL (University College London).

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Abstract

Salamanders such as the red spotted newt Notophthalamus viridescens and the axolotl Ambystoma mexicanum regenerate a number of anatomical structures following injury. Prod1 is believed to guide patterning processes operating during limb regeneration, however the molecular mechanism through which it operates is unclear. Being glycosylphosphatidylinositol (GPI) anchored, Prod1 does not make direct contact with the cytoplasm, raising questions as to how it functions in the transfer of information across the cell membrane. The transmembrane epidermal growth factor receptor was shown to associate with Prod1, initiating MAPK signalling and resulting in the induction of matrix metalloprotease 9 expression (MMP9). MMP9 is known to be rapidly upregulated in the hours following amputation in the wound epithelium, a structure essential for regeneration formed by the migration of epidermal cells across the surface of the amputation plane. Patches of newt limb skin explanted into culture were used as a model for this process. A sheet of cells expressing MMP9 was seen to migrate out from skin patches, and this was shown to be sensitive to MMP inhibitors. Further to this, upregulation of MMP9 was seen to occur in the dermis of explanted skin patches, a layer of the skin known to be instructive to the patterning of the limb. The relationship of Prod1s structure to its MMP9 inducing function was investigated through the creation of a series of point mutants, and it was shown that amino acids located on the α-helix of the protein were essential for this function. Axolotl Prod1 lacks a GPI anchor, however despite the requirement of newt Prod1 for GPI anchorage in order to induce MMP9 expression in either newt or axolotl cells, axolotl Prod1 was fully functional in cells from either species. There was some indication that amino acids on the α-helix may confer this ability to axolotl Prod1.

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