Lenalidomide and other immunomodulatory drugs (IMiDs) are highly effective treatments for multiple myeloma, myelodysplastic syndrome with del(5q), and other hematologic neoplasms. IMiDs were recently shown to act by a novel drug mechanism-modulation of an E3 ubiquitin ligase to specifically ubiquitinate target proteins. IMiDs bind cereblon (CRBN), the substrate adaptor for the CRBN-CRL4 E3 ubiquitin ligase, and induce the ubiquitination of two lymphoid transcription factors by this complex. These factors are essential for the survival of multiple myeloma cells, so their subsequent proteasomal degradation explains the efficacy of IMiDs in this condition. Activation of an ubiquitin ligase to specifically target proteins for degradation is a completely novel drug mechanism with therapeutic implications for many clinical conditions. However, further understanding of the interactions between CRBN and lenalidomide-dependent substrates is needed before we can apply this mechanism more broadly. Specifically, we must understand whether lenalidomide allosterically increases the overall activity of CRBN-CRL4 on its endogenous substrates or bridges the CRBN-substrate interface to recruit target proteins to CRBN. I will combine unbiased and targeted mutagenesis of CRBN to investigate the hypothesis that lenalidomide works by bridging interactions between CRBN and recruited substrates. In Aim 1, I will use scanning mutagenesis to identify the entire landscape of CRBN residues essential for lenalidomide-induced substrate degradation. In this approach, each residue of CRBN is mutated to all possible amino acids, one position at a time. CRBN mutations which abrogate lenalidomide-induced substrate degradation will be mapped onto the crystal structure of the CRBN-IMiD complex to identify residues essential for interactions with lenalidomide, recruited substrates, and other CRBN-CRL4 complex members. In Aim 2, I will evaluate the effects of targeted mutations of CRBN residues adjacent to the IMiD binding site. If lenalidomide works by a bridging mechanism, it should be possible to identify mutations in this region which affect substrate recruitment without affecting drug binding. Specifically, I will test the effects of increasing the steric bulk of four non-polar amino acids adjacent to the IMiD-binding pocket on lenalidomide-induced substrate degradation, substrate recruitment, and lenalidomide binding. I will also test the effects of mutating four charged residues surrounding the IMiD binding site to alanine. Through hypothesis driven and unbiased approaches, this work will thoroughly investigate the interactions between CRBN and lenalidomide-induced substrates. Understanding these interactions is key to applying this novel drug mechanism to other molecular targets and clinical conditions.