Transmissible spongiform encephalopathies have been observed exclusively in organisms expressing the host-encoded prion protein (PrP). The function of the cellular isoform of PrP found in healthy organisms has so far not been identified, although there are indications of a role in signal transduction in neurons. To gain further insight into the functional properties of cellular PrP, this paper investigated the binding of the C-terminal SH3 domain of the murine growth factor receptor-bound protein 2 (Grb2) to the murine PrP, using NMR, fluorescence, and circular dichroism spectroscopy. The SH3-binding site in murine PrP was thus found to be in the highly conserved region of residues 100−109, which contains prolines in positions 101 and 104. The protein−protein interaction, with a K_D value of 5.5 μM, is abolished when either of these two prolines is replaced by leucine. In humans, two corresponding Pro → Leu exchanges are found in patients who present with the Gerstmann−Sträussler−Scheinker syndrome. The results of the present study thus indicate a possible mechanism by which amino acid exchanges could influence a specific protein−protein interaction in a complex signal transduction cascade, which might be of functional significance in health and disease.