Molecular interactions of alpha(2)-adrenergic receptor peptides with G proteins.

Abstract

A variety of hormones and growth factors transmit signals to cells by binding to receptors and initiating a cascade of events within the cell, leading to a cellular response. Certain plasma membrane receptors (including the $\alpha\sb2$-adrenergic receptor) are coupled to cytoplasmic effector enzymes through heterotrimeric guanine nucleotide binding proteins (G-proteins). The overall goal of my thesis project is to develop a structural map of the interactions between receptor and G-protein. We used synthetic peptides derived from specific sequences in the $\alpha\sb2$AR to determine the critical regions involved in receptor-G protein interactions. Previous work determined that a receptor-derived peptide from the 3$\sp{\rm rd}$ intracellular loop (peptide Q) can antagonize G protein interactions and directly activate G protein. This thesis focuses on determining the specific residues in the G protein to which this receptor-derived peptide binds. To this end, I developed and characterized a photo-affinity probe of peptide Q using a heterobifunctional diazopyruvoyl photoaffinity cross-linking agent. Upon photolysis of the modified peptide with G protein, I observed specific, competable labelling of sites on both the $\alpha$ and $\beta$ subunits but not the $\gamma$ subunit of G-protein. I also show that the $\beta\gamma$ subunit is required for the modified peptide to activate G-protein, indicating a functional significance for the $\beta$ subunit interaction. The regions on the $\alpha$ and $\beta$ subunit to which this peptide binds were mapped by protease digestion of the cross-linked G protein followed by gel electrophoresis or high performance liquid chromatography purification and mass spectroscopy of the proteolysis fragments. Using these techniques, I determined that peptide Q binds to the 2 kD amino-terminal fragment of $\alpha\sb{\rm o}$ and a site within the carboxy-terminal 60 amino acids of the $\beta$ subunit. This represents the first report of the interaction site of the $\beta\gamma$ subunit with a receptor-like G protein activator. Binding of the activator peptide to the amino-terminus of $\alpha$ is consistent with a number of reports indicating that the amino-terminus of $\alpha$ interacts with receptors. Identification of these binding sites could aid in the development of therapeutic agents which could target the receptor-G protein interface.