Differential Localization of G Protein βγ Subunits

Abstract

G protein βγ subunits play essential roles in regulating cellular signaling cascades, yet little is known about their distribution in tissues or their subcellular localization. While previous studies have suggested specific isoforms may exhibit a wide range of distributions throughout the central nervous system, a thorough investigation of the expression patterns of both Gβ and Gγ isoforms within subcellular fractions has not been conducted. To address this, we applied a targeted proteomics approach known as multiple-reaction monitoring to analyze localization patterns of Gβ and Gγ isoforms in pre- and postsynaptic fractions isolated from cortex, cerebellum, hippocampus, and striatum. Particular Gβ and Gγ subunits were found to exhibit distinct regional and subcellular localization patterns throughout the brain. Significant differences in subcellular localization between pre- and postsynaptic fractions were observed within the striatum for most Gβ and Gγ isoforms, while others exhibited completely unique expression patterns in all four brain regions examined. Such differences are a prerequisite for understanding roles of individual subunits in regulating specific signaling pathways throughout the central nervous system.

Quantitative Multiple-Reaction Monitoring Proteomic Analysis of Gβ and Gγ Subunits in C57Bl6/J Brain Synaptosomes

“…Two proteotypic peptides were selected per protein (see Material and Method for more detail) to increase the accuracy of quantification. No proteotypic peptides were selected for Gγ 5 and Gγ 11 as we were unable to identify a correct peak of sufficient signal intensity to pass acceptable signal to noise criteria in a previous study 62 .…”

“…To overcome this issue, we previously developed a targeted multiple reaction monitoring (MRM) 64, 65 mass spectrometric approach to identify neuronal Gβ and Gγ subunits. We found a regional and subcellular expression pattern of four Gβ (Gβ 1 , Gβ 2 , Gβ 4 , and Gβ 5 ) and six Gγ (Gγ 2 , Gγ 3 , Gγ 4 , Gγ 7 , Gγ 12 , and Gγ 13 ) subunits in mouse crude synaptosomes of cortex, cerebellum, hippocampus, and striatum 62 . However, we were unable to quantify and compare the expression level of each Gβ and Gγ subunit.…”

“…Crude synaptosomes were made from adult, male C57Bl6/J mice as described previously 62, 67, 68 . Briefly, whole brains were homogenized in 20 mL of a 0.32 M sucrose solution (0.32M sucrose, 4.2 mM HEPES pH 7.4, 0.1 mM CaCl 2 , 1 mM MgCl 2 , 1.54 µM aprotinin, 10.7 µM leupeptin, 0.95 µM pepstatin, and 200 µM PMSF).…”

GβγBinds to the Extreme C Terminus of SNAP25 to Mediate the Action of Gi/o-Coupled G Protein–Coupled Receptors

“…There are five Gb subunits and 12 Gg subunits in the human genome, indicating a high degree of redundancy, making knockout or mutagenesis of Gbg subunits unfeasible . Although studies have been conducted pertaining to the distribution of Gb and Gg subunits in the brain (Betke et al, 2014), it is not currently known whether a specific combination of subunits is responsible for the Gbg-SNARE interaction. The possibility of multiple effectors for any given Gbg would also be a confounding factor in such a knockout.…”

Applications of targeted proteomics in systems biology and translational medicine

“…In developmental biology, Betke et al. examined the differential localization and abundance of G‐beta and G‐gamma isoforms of G proteins in pre‐ and post‐synaptic fractions isolated from the cortex, cerebellum, hippocampus, and striatum of adult C57Bl6/J mice which showed significant differences in subcellular localization of different isoforms and provided an advance in understanding the roles of various subunits in different brain tissues 44. Pharmacological and toxicological examinations of 27 cytochrome P450 proteins in Balb/c mouse liver microsomes and tissue lysates from kidney, lung, intestine, heart, and brain across different developmental stages, including pregnancy were performed by Hersmann and colleagues 45.…”