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Conversion of integral membrane receptors into soluble forms

Final Report Summary - GPCRS (Conversion of integral membrane receptors into soluble forms)

G-protein coupled receptors (GPCRs) are cell surface receptors that mediate the cellular responses to an enormous diversity of endogenous signaling molecules such as hormones and neurotransmitters, as well as environmental signals such as pheromones, smells, taste and light. GPCRs are a major target for the pharmaceutical industry as is reflected by the fact that more than 40% of all prescription drugs available today act on a GPCR and represent about a quarter of the top-selling drugs worldwide, with annual revenues in excess of 30€ billion. However, effective drug design and functional characterization of these receptors is strongly limited by the absence of sufficient high-resolution structural information. Solving crystal structures of more members of the GPCR family in ligand-free forms and in the complexes with agonist and/or antagonist would qualitatively and quantitatively significantly improve drug-design procedures and lead to detailed understanding of the activation process catalyzed by GPCR.
Low expression levels, toxicity to cells, inability to refold from solubilized non-native aggregates in vitro, difficulties in purification, limited stability in detergent-solubilized form, and heterogeneity of detergent-solubilized form, i.e. properties affected mainly by low solubility, are major problems in effort of crystallization and solving structure of GPCRs and integral membrane proteins in general. In principle, all these obstacles can be overcome by improving the solubility of the receptors, or in the extreme case by converting the receptors from “membrane-soluble” into water-soluble form.
In this project, we proposed to convert one member of GPCR family - κ-opioid receptor- into a water-soluble form. This is intended to be an experimental proof of feasibility of similar projects in the future. Redesigning the entire membrane embedded surface of a GPCR by substituting the hydrophobic amino acids of the protein/lipid interface with suitable polar or charged residues to produce a molecule that is able to fold and function in aqueous solution represents an ambitious protein-engineering problem of high combinatorial complexity. It was highly improbable that the desired result can be reached in a single step by rational design. Instead, we have chosen a highly interdisciplinary approach that combines the strengths of computational and experimental tools, of design, selection and in vitro evolution. The strategy we proposed to use relies on the proven expertise of the Plückthun group in the rational design of protein libraries. From these libraries, functional molecules can efficiently be selected by ribosome display methods. Selected sequences can be further optimized using the techniques of directed in vitro evolution.
We have chosen the κ-opioid receptor that belongs to the more intensively studied rhodopsin-like subgroup of the GPCR family. The receptor plays an important role in modulation of pain perception, mood regulation, stress response, depression, appetite, circadian rhythm, temperature regulation, and has high therapeutic potential in the treatment of certain drug addiction.