Mechanisms of GPCR Signaling

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The light-activated visual receptor rhodopsin has provided the foundation for understanding the structure and mechanism of G protein-coupled receptors (GPCRs). Nevertheless, there remain fundamental unanswered questions about how these receptors work. Here, we target several basic questions that are relevant for understanding their mechanism(s) of activation. The approach is primarily through structural measurements using solid-state NMR spectroscopy. The existing crystal structures of these receptors provide a high- resolution framework to study in detail the role of specific residues and motifs in receptor activation. Because of the high conservation of residues between the visual and ligand-activated GPCRs, the emerging consensus is that rather than being unique, the visual receptors provide a basis for understanding the common structural and dynamic elements in these receptors. The general experimental strategy is to use solid-state NMR spectroscopy in combination with mutational, optical and biochemical methods to target specific regions in the inactive and active states of the dim-light receptor, rhodopsin. The goal is to understand in atomic detail the interplay between specific signature, group-conserved and subfamily-conserved motifs in the activation mechanism of rhodopsin and establish a common basis for the activation of other GPCRs. Four specific aims address structure-function questions involving regions on the extracellular side of rhodopsin (Aim 1) and within the transmembrane (TM) core and on the intracellular side of the receptor (Aim 2). In Aim 1, we will establish the role of Trp6.48 – a key residue that mediates retinal isomerization and Schiff base deprotonation with the conserved TM core of the receptor. In Aim 2, we address how retinal Schiff base deprotonation leads to activation. The working model is that there are two triggers, one electrostatic and one steric in nature. We target the conserved TM core of rhodopsin composed of interlocking signature and group- conserved residues. The working model is that the TM core is composed of two packing clusters and two activation switches. These provide stable and flexible elements to the receptor, respectively. In Aim 3, we focus on the G protein and its interactions with residues on the intracellular surface of the active Meta II intermediate. In this aim, we address the role of the membrane environment in receptor stability and activation. Finally, in Aim 4 we use the information garnered above and from past studies to determine the basis for two retinal diseases, congenital stationary night blindness and autosomal dominant retinitis pigmentosa.