Heidi E. Hamm

The Many Faces of G Protein Signaling

A large number of hormones, neurotransmitters, chemokines, local mediators, and sensory stimuli exert their effects on cells and organisms by binding to G protein-coupled receptors. More than a thousand such receptors are known, and more are being discovered all the time. Heterotrimeric G proteins transduce ligand binding to these receptors into intracellular responses, which underlie physiological responses of tissues and organisms. G proteins play important roles in determining the specificity and temporal characteristics of the cellular responses to signals. They are made up of a, b, and g subunits, and although there are many gene products encoding each subunit (20 a, 6 b, and 12 g gene products are known), four main classes of G proteins can be distinguished: Gs, which activates adenylyl cyclase; Gi, which inhibits adenylyl cyclase; Gq, which activates phospholipase C; and G12 and G13, of unknown function. G proteins are inactive in the GDP-bound, heterotrimeric state and are activated by receptor-catalyzed guanine nucleotide exchange resulting in GTP binding to the a subunit. GTP binding leads to dissociation of GazGTP from Gbg subunits and activation of downstream effectors by both GazGTP and free Gbg subunits. G protein deactivation is rate-limiting for turnoff of the cellular response and occurs when the Ga subunit hydrolyzes GTP to GDP. The recent resolution of crystal structures of heterotrimeric G proteins in inactive and active conformations provides a structural framework for understanding their role as conformational switches in signaling pathways. As more and more novel pathways that use G proteins emerge, recognition of the diversity of regulatory mechanisms of G protein signaling is also increasing. The recent progress in the structure, mechanisms, and regulation of G protein signaling pathways is the subject of this review. Because of space considerations, I will concentrate mainly on recent studies; readers are directed to a number of excellent reviews that cover earlier studies.

Heterotrimeric G protein activation by G-protein-coupled receptors

Heterotrimeric G proteins have a crucial role as molecular switches in signal transduction pathways mediated by G-protein-coupled receptors. Extracellular stimuli activate these receptors, which then catalyse GTP–GDP exchange on the G protein α-subunit. The complex series of interactions and conformational changes that connect agonist binding to G protein activation raise various interesting questions about the structure, biomechanics, kinetics and specificity of signal transduction across the plasma membrane.

Molecular determinants of selectivity in 5-hydroxytryptamine1B receptor-G protein interactions.

The recognition between G protein and cognate receptor plays a key role in specific cellular responses to environmental stimuli. Here we explore specificity in receptor-G protein coupling by taking advantage of the ability of the 5-hydroxytryptamine1B (5-HT1B) receptor to discriminate between G protein heterotrimers containing Gαi1 or Gαt. Gi1 can interact with the 5-HT1B receptor and stabilize a high affinity agonist binding state of this receptor, but Gt cannot. A series of Gαt/Gαi1 chimeric proteins have been generated in Escherichia coli, and their functional integrity has been reported previously (Skiba, N. P., Bae, H., and Hamm, H. E. (1996) J. Biol. Chem. 271, 413–424). We have tested the functional coupling abilities of the Gαt/Gαi1 chimeras to 5-HT1Breceptors using high affinity agonist binding and receptor-stimulated guanosine 5′-3-O-(thio)triphosphate (GTPγS) binding. In the presence of βγ subunits, amino acid residues 299–318 of Gαi1 increase agonist binding to the 5-HT1Breceptor and receptor stimulation of GTPγS binding. Moreover, Gαi1 containing only Gαt amino acid sequences from this region does not show any coupling ability to 5-HT1B receptors. Our studies suggest that the α4 helix and α4-β6 loop region of Gαs are an important region for specific recognition between receptors and Gi family members.

How activated receptors couple to G proteins.

G protein-coupled receptors (GPCRs) are involved in the control of every aspect of our behavior and physiology. This is the largest class of receptors, with several hundred GPCRs identified thus far. Examples are receptors for hormones such as calcitonin and luteinizing hormone or neurotransmitters such as serotonin and dopamine. G protein-coupled receptors can be involved in pathological processes as well and are linked to numerous diseases, including cardiovascular and mental disorders, retinal degeneration, cancer, and AIDS. More than half of all drugs target GPCRs and either activate or inactivate them. Binding of specific ligands, such as hormones, neurotransmitters, chemokines, lipids, and glycoproteins, activates GPCRs by inducing or stabilizing a new conformation in the receptor (1, 2). Activated receptors (R*) can then activate heterotrimeric G proteins (composed of α.GDP, β, and γ subunits) on the inner surface of the cell membrane (3–5).

Structural basis of function in heterotrimeric G proteins

Heterotrimeric guanine-nucleotide-binding proteins (G proteins) act as molecular switches in signaling pathways by coupling the activation of heptahelical receptors at the cell surface to intracellular responses. In the resting state, the G-protein alpha subunit (Galpha) binds GDP and Gbetagamma. Receptors activate G proteins by catalyzing GTP for GDP exchange on Galpha, leading to a structural change in the Galpha(GTP) and Gbetagamma subunits that allows the activation of a variety of downstream effector proteins. The G protein returns to the resting conformation following GTP hydrolysis and subunit re-association. As the G-protein cycle progresses, the Galpha subunit traverses through a series of conformational changes. Crystallographic studies of G proteins in many of these conformations have provided substantial insight into the structures of these proteins, the GTP-induced structural changes in Galpha, how these changes may lead to subunit dissociation and allow Galpha and Gbetagamma to activate effector proteins, as well as the mechanism of GTP hydrolysis. However, relatively little is known about the receptor-G protein complex and how this interaction leads to GDP release from Galpha. This article reviews the structural determinants of the function of heterotrimeric G proteins in mammalian systems at each point in the G-protein cycle with special emphasis on the mechanism of receptor-mediated G-protein activation. The receptor-G protein complex has proven to be a difficult target for crystallography, and several biophysical and computational approaches are discussed that complement the currently available structural information to improve models of this interaction. Additionally, these approaches enable the study of G-protein dynamics in solution, which is becoming an increasingly appreciated component of all aspects of G-protein signaling.

RGS4-dependent attenuation of M4 autoreceptor function in striatal cholinergic interneurons following dopamine depletion.

Parkinson disease is a neurodegenerative disorder whose symptoms are caused by the loss of dopaminergic neurons innervating the striatum. As striatal dopamine levels fall, striatal acetylcholine release rises, exacerbating motor symptoms. This adaptation is commonly attributed to the loss of interneuronal regulation by inhibitory D2 dopamine receptors. Our results point to a completely different, new mechanism. After striatal dopamine depletion, D2 dopamine receptor modulation of calcium (Ca2+) channels controlling vesicular acetylcholine release in interneurons was unchanged, but M4 muscarinic autoreceptor coupling to these same channels was markedly attenuated. This adaptation was attributable to the upregulation of RGS4—an autoreceptor-associated, GTPase-accelerating protein. This specific signaling adaptation extended to a broader loss of autoreceptor control of interneuron spiking. These observations suggest that RGS4-dependent attenuation of interneuronal autoreceptor signaling is a major factor in the elevation of striatal acetylcholine release in Parkinson disease.

Antagonists of the Receptor-G Protein Interface Block Gi-coupled Signal Transduction

The carboxyl terminus of heterotrimeric G protein α subunits plays an important role in receptor interaction. We demonstrate that peptides corresponding to the last 11 residues of Gαi1/2 or Gαo1 impair agonist binding to A1 adenosine receptors, whereas Gαs or Gαt peptides have no effect. Previously, by using a combinatorial library we identified a series of Gαtpeptide analogs that bind rhodopsin with high affinity (Martin, E. L., Rens-Domiano, S., Schatz, P. J., and Hamm, H. E. (1996)J. Biol. Chem. 271, 361–366). Native Gαi1/2 peptide as well as several analogs were tested for their ability to modulate agonist binding or antagonist-agonist competition using cells overexpressing human A1 adenosine receptors. Three peptide analogs decreased the K i , suggesting that they disrupt the high affinity receptor-G protein interaction and stabilize an intermediate affinity state. To study the ability of the peptides to compete with endogenous Gαiproteins and block signal transduction in a native setting, we measured activation of G protein-coupled K+ channels through A1 adenosine or γ-aminobutyric acid, type B, receptors in hippocampal CA1 pyramidal neurons. Native Gαi1/2, peptide, and certain analog peptides inhibited receptor-mediated K+ channel gating, dependent on which receptor was activated. This differential perturbation of receptor-G protein interaction suggests that receptors that act on the same G protein can be selectively disrupted.

Structural and functional relationships of heterotrimeric G-proteins.

Heterotrimeric GTP‐binding proteins (G‐proteins) are a critical component of signal transduction pathways that carry information received at the cell surface to the appropriate cellular effector system, ultimately achieving a specific cellular response. Heterotrimeric G‐proteins consist of an a‐subunit, which contains the guanine nucleotide binding site and intrinsic GTPase activity, and an inseparable βγ‐subunit complex. G‐proteins act to define the specificity by which a receptor regulates a particular intracellular signaling system, as well as to regulate the duration of the signal. A great deal of structural and functional insight into how G‐protein‐mediated signal transduction occurs has recently been achieved. This review will discuss the structural features of G‐proteins, as well as detail the mechanism by which G‐proteins interact with receptors and effectors.—Rens‐Domiano, S., Hamm, H. E. Structure and functional relationships of heterotrimeric G‐proteins. FASEBJ. 9, 1059‐1066 (1995)

Gβγ acts at the C terminus of SNAP-25 to mediate presynaptic inhibition

Presynaptic inhibition mediated by G protein–coupled receptors may involve a direct interaction between G proteins and the vesicle fusion machinery. The molecular target of this pathway is unknown. We demonstrate that Gβγ-mediated presynaptic inhibition in lamprey central synapses occurs downstream from voltage-gated Ca2+ channels. Using presynaptic microinjections of botulinum toxins (BoNTs) during paired recordings, we find that cleavage of synaptobrevin in unprimed vesicles leads to an eventual exhaustion of synaptic transmission but does not prevent Gβγ-mediated inhibition. In contrast, cleavage of the C-terminal nine amino acids of the 25 kDa synaptosome-associated protein (SNAP-25) by BoNT A prevents Gβγ-mediated inhibition. Moreover, a peptide containing the region of SNAP-25 cleaved by BoNT A blocks the Gβγ inhibitory effect. Finally, removal of the last nine amino acids of the C-terminus of SNAP-25 weakens Gβγ interactions with soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complexes. Thus, the C terminus of SNAP-25, which links synaptotagmin I to the SNARE complex, may represent a target of Gβγ for presynaptic inhibition.

Mechanism of the receptor-catalyzed activation of heterotrimeric G proteins

Heptahelical receptors activate intracellular signaling pathways by catalyzing GTP for GDP exchange on the heterotrimeric G protein α subunit (Gα). Despite the crucial role of this process in cell signaling, little is known about the mechanism of G protein activation. Here we explore the structural basis for receptor-mediated GDP release using electron paramagnetic resonance spectroscopy. Binding to the activated receptor (R*) causes an apparent rigid-body movement of the α5 helix of Gα that would perturb GDP binding at the β6-α5 loop. This movement was not observed when a flexible loop was inserted between the α5 helix and the R*-binding C terminus, which uncouples R* binding from nucleotide exchange, suggesting that this movement is necessary for GDP release. These data provide the first direct observation of R*-mediated conformational changes in G proteins and define the structural basis for GDP release from Gα.