Structure and Function of Signal Transduing Components

Icahn School Of Medicine At Mount Sinai, New York NY

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Abstract

DESCRIPTION (provided by applicant): The long-term goal of this research project is to develop a detailed molecular understanding of how signal is propagated through heterotrimeric G protein pathways. Many hormones, neurotransmitters, autocrine and paracrine factors function through receptors that use hetrotrimeric G proteins as transducers. We are particularly interested in the mechanisms of signal transfer from the G protein subunits to their effectors adenylyl cyclase and phospholipase C-P2. Multiple regions within the G protein subunits interact with multiple regions on the effector surface. Studies in the previous term had focused on identifying the functions of these contacts. For Ga interactions with phospholipaseC-a2, we have found that the contact sites spread over five of the seven blades of Ga can be divided into two categories, signal transfer regions that are capable of effector activation and general binding domains that interact with the effector and contribute to overall binding affinity but do not directly participate in signal propagation. Stimulation of phospholipase C-beta2 by Gbetagamma involves two signal transfer regions and three general binding domains on Ga and there appears to be cooperativity between the two signal transfer regions. Similarly Gas stimulation of adenylyl cyclase involves multiple sites of contact including the Switch II helix that functions as a signal transfer region and the alpha3-beta5 loop that functions as a general binding domain on Gas. In the coming term we will use a combinatorial approach combined with NMR spectroscopy studies to analyze in detail the signal transfer regions and general binding domains to understand the design of these regions. For this we will use peptide-on-plasmid libraries to analyze the interactions between the Ga and phospholipase C-beta2 and adenylyl cyclase 2. We will also study Galphas interactions with adenylyl cyclases 2 and 6. In the latter case we will conduct combinatorial analysis of both the signal transfer region on Gas and the signal receiving regions of adenylyl cyclase. The conclusions from the peptide experiments will be confirmed by mutational analysis of the G protein subunits and the effectors. The functional studies will be complemented by NMR studies to understand the structural basis of the observed functional effects. These experiments should allow for the design of compounds that function as agonists and antagonists at these intracellular sites and have the potential to function as therapeutic agents in many diseases including immune disorders, diabetes, and metabolic disorders.