Engineering GPCR signaling pathways with RASSLs

Abstract

We are creating families of designer G protein-coupled receptors (GPCRs) to allow for precise spatiotemporal control of GPCR signaling in vivo. These engineered GPCRs, called receptors activated solely by synthetic ligands (RASSLs), are unresponsive to endogenous ligands but can be activated by nanomolar concentrations of pharmacologically inert, drug-like small molecules. Currently, RASSLs exist for the three major GPCR signaling pathways (G(s), G(i) and G(q)). We review these advances here to facilitate the use of these powerful and diverse tools.

Figure 1

Creating RASSLs by targeted mutagenesis. A conserved residue(s) in the canonical binding pocket of biogenic amine receptors (e.g., adrenergic, serotonin, histamine) is mutated to eliminate the binding and activation of the receptor for the native ligand. A model using the coordinates of the ••-adrenergic receptor structure is used to illustrate this. In all biogenic amine GPCRs, the binding pocket is composed of a conserved aspartic acid (D113^3.32 in the •2-AR model; SIDE VIEW) and conserved aromatic and polar residues (TOP VIEW). Mutation of the highly conserved Asp to Ser (D113S) renders the •2-AR insensitive to •-AR exogenous and endogenous agonists, such as isoproterenol, epinephrine, and norepinephrine. However, the D113S mutant receptor could be activated by the synthetic ligand L-185, 870. Such a targeted mutagenesis approach was used to create both peptidergic RASSLs (e.g., •-opioid, MC4-melanocortin) and nonpeptidergic RASSLs (H1-histamine, 5-HT2A serotonin, 5-HT4 serotonin, •2-adrenergic) (see text for details).

Figure 2

Myriad ligand-dependent and -independent phenotypes are induced by tissue-specific expression of a single RASSL. Ligand-induced phenotypes are noted in red; constitutive signaling-induced phenotypes are in blue. Asterisks indicate tissues in which RO1 expression results in embryonic or perinatal lethality. Conditional expression allows RASSL researchers to avoid embryonic lethality and analyze adult phenotypes.

Figure 3

Directed molecular evolution to create novel RASSLs. Shown is a generic scheme for obtaining RASSLs via directed molecular evolution. In brief, a large library of randomly mutated GPCRs is obtained by error-prone PCR and used to efficiently transform the appropriate yeast strain. Yeast with functional GPCRs is grown in histidine-deficient medium in the presence of the inert ligand (in this case clozapine-N-oxide, CNO), and surviving colonies are expanded and characterized pharmacologically. GPCRs with the appropriate pharmacological profiles are subjected to iterative rounds of additional mutagenesis and selection until the ideal RASSL is obtained. Candidate RASSLs are subjected to growth assays in the presence and absence of candidate ligands to screen out those with elevated levels of constitutive activity. Typically several candidate inert ligands are used in the initial screens to determine which are most suitable for directed molecular evolution and then one or more chosen for further testing. The final choice of the candidate ligand is based on its potency and drug-like properties.