Introduction

G Protein-coupled Receptors and RGS Proteins

Adele Stewart2; Rory A. Fisher1    Department of Pharmacology, University of Iowa Carver College of Medicine, Iowa City, Iowa, USA
1 Corresponding author: email address: rory-fisher@uiowa.edu
2 Present address: Department of Pharmacology, Vanderbilt University, Nashville, Tennessee, USA

1 GPCR Physiology, Pathophysiology, and Pharmacology

G protein-coupled receptors (GPCRs) represent the largest class of cell surface receptors and are responsible for transducing extracellular signals in the form of peptides, neurotransmitters, hormones, odorants, light, ions, nucleotides, or amino acids into the intracellular environment. It is now believed that the GPCR superfamily contains over 1000 genes in humans, comprising ~ 2% of all gene-encoding DNA.1,2 Given the diversity of GCPR stimuli and the abundance of GPCR-encoding genes in the human genome, it is not surprising that GPCR dysregulation plays a causative role in many human maladies including cardiovascular diseases, neuropsychiatric disorders, metabolic syndromes, carcinogenesis, and viral infections.3–6 In fact, it is estimated that 40–50% of currently marketed pharmaceuticals target GPCRs, arguably the most remunerative drug class with worldwide sales totaling $47 billion in 2003.3

Though new GPCR-targeted drugs are in the pharmaceutical industry pipeline,7 a number of challenges have emerged in the development of novel therapeutics aimed at disrupting or enhancing signaling through GPCRs. In particular, for many years, a lack of high-resolution crystal structures made in silico bioinformatic drug screening challenging. The recently solved structure of the β2-adrenergic receptor in complex with Gαs8 (amongst others) will likely facilitate such efforts in the coming years. Additional hurdles in GPCR drug development include agonist-induced receptor desensitization and tolerance; activation or inhibition of multiple GPCR effector cascades; a lack of selectivity between ligand-specific receptor subtypes; and the possibility of off-target effects due to receptor expression in multiple cells, tissues or organs in the body.7 Though receptor targeting is ideal due to the lack of need for intracellular drug trafficking, it is now believed that GPCR effectors and regulators may also be viable drug targets and might represent a means to improve therapeutic efficacy and specificity.

2 GPCR Signal Transduction: Heterotrimeric G Proteins

Structurally, GPCRs are characterized by seven membrane-spanning alpha helices with an extracellular N-terminal tail, often, but not exclusively, involved in ligand binding, and intracellular loops and a C-terminus involved in guanine-nucleotide regulatory protein (G protein) coupling and receptor regulation. Ligand binding is believed to induce a conformational change in the receptor that promotes G protein association.9 Activated receptors function as guanine nucleotide exchange factors (GEFs) for the α subunit of the heterotrimeric G protein complex. Gα will then transition from its inactive guanosine diphosphate (GDP)-bound form to the active guanosine triphosphate (GTP)-bound monomer, dissociating from the Gβγ dimer (Fig. 1). There are four families of Gα subunits in mammals (Gαs, Gαi, Gαq, and Gα12/13), which differ in their specific effector coupling, downstream signaling, and net cellular response. GPCR coupling to Gα subunits is highly selective allowing for ligand-specific modulation of downstream signaling in cells. Gα subunits contain two characterized functional domains: a GTP-binding cassette homologous to that found in Ras-like small GTPases and a helical insertion. GCPRs trigger a conformational change in the three flexible “switch” regions of the GTP-binding domain. The helical insertion, conversely, is unique to heterotrimeric G proteins and functions to sequester the guanine nucleotide in the GTP-binding domain. Nucleotide dissociation requires displacement of this structure, a process facilitated by active GPCRs.10,11 Both GTP-bound Gα and Gβγ activate effector molecules, which include enzymes, ion channels, and protein kinases.3 Deactivation of G-protein signaling occurs by the intrinsic hydrolysis of GTP to GDP by the Gα subunit, which occurs at a rate that varies among the G-protein subfamilies.12

Five genes encode Gβ subunits and twelve genes encode the varying Gγ isoforms resulting in an impressive diversity of possible dimeric Gβγ complexes.13 Gβ and Gγ subunits form obligate heterodimers in vivo as Gβ requires Gγ for proper protein folding.14 Gγ proteins have a simple structure containing two α-helices joined by a linker loop, which form a coiled-coil interaction with the N-terminal α-helix of Gβ.15 The remainder of the Gβ subunit consists of a β-propeller motif composed of tryptophan-aspartic acid (WD) repeats forming arrangements of antiparallel β sheets. Crystal structures of effector-bound Gβγ complexes have revealed that this β-propeller structure is intimately involved in effector coupling.16,17 Unsurprisingly, this effector-binding site largely overlaps with the region responsible for interaction between Gβγ dimers and the switch II region of Gα, which explains the lack of Gβγ signaling when sequestered in the heterotrimeric G protein complex.12 It is known that some Gβ and Gγ subunits preferentially interact18–20 leading to the supposition that there may be some selectivity in Gβγ dimer receptor/G protein coupling and effector activation. Indeed, studies in individual Gβ and Gγ knockout models have revealed unique phenotypic consequences for loss of specific subunits implying that these proteins are not as interchangeable as was originally believed.21

3 G Protein Regulation

Regulation of GPCRs is complex with multiple layers of interconnected signaling pathways activated upon receptor simulation that feedback to impact receptor function. The best characterized GPCR regulatory mechanisms are mediated by G protein-coupled receptor kinases (GRKs), arrestins, and regulator of G protein-signaling (RGS) proteins. The Gβγ dimer facilitates membrane targeting of GRKs resulting in GRK-mediated GPCR phosphorylation. This modification recruits β-arrestins, which sterically hinder further G-protein coupling to the receptor.22 Though their role in GPCR desensitization has been well characterized, it is now appreciated that arrestins are multifunctional scaffolds...