Receptor-effector coupling by G proteins

The primary structure of G proteins as deduced from purified proteins and cloned subunits is presented. When known, their functions are discussed, as are recent data on direct regulation of ionic channels by G proteins. Experiments on expression of alpha subunits, either in bacteria or by in vitro translation of mRNA synthesized from cDNA are presented as tools for definitive assignment of function to a given G protein. The dynamics of G protein-mediated signal transduction are discussed. Key points include the existence of two superimposed regulatory cycles in which upon activation by GTP, G proteins dissociate into alpha and beta gamma and their dissociated alpha subunits hydrolyze GTP. The action of receptors to catalyze rather than regulate by allostery the activation of G proteins by GTP is emphasized, as is the role of subunit dissociation, without which receptors could not act as catalysts. To facilitate the reading of this review, we have presented the various subtopics of this rapidly expanding field in sections 1-1X, each of which is organized as a self-contained sub-chapter that can be read independently of the others.     

How regulators of G protein signaling achieve selective regulation

The regulators of G protein signaling (RGS) are a family of cellular proteins that play an essential regulatory role in G protein-mediated signal transduction. There are multiple RGS subfamilies consisting of over 20 different RGS proteins. They are basically the guanosine triphosphatase (GTPase)-accelerating proteins that specifically interact with G protein alpha subunits. RGS proteins display remarkable selectivity and specificity in their regulation of receptors, ion channels, and other G protein-mediated physiological events. The molecular and cellular mechanisms underlying such selectivity are complex and cooperate at many different levels. Recent research data have provided strong evidence that the spatiotemporal-specific expression of RGS proteins and their target components, as well as the specific protein-protein recognition and interaction through their characteristic structural domains and functional motifs, are determinants for RGS selectivity and specificity. Other molecular mechanisms, such as alternative splicing and scaffold proteins, also significantly contribute to RGS selectivity. To pursue a thorough understanding of the mechanisms of RGS selective regulation will be of great significance for the advancement of our knowledge of molecular and cellular signal transduction.     

The importance of G-protein beta lambda subunits

As the properties of more and more isoforms of the molecules involved in G-protein-mediated signal transduction pathways are unravelled, surprising diversity and versatility are being revealed. The path from receptor to effector is not dictated exclusively by the alpha subunits of heterotrimetric G proteins. The nature of the beta lambda subunit complex probably controls interactions of G(alpha) with receptors. In addition, dissociation of G(alpha)-GTP from G(beta lambda)provides two signalling complexes, and these proteins regulate effectors independently or synergistically. Synergistic or conditional regulation of effectors by G(alpha) and G(beta lambda)can provide a molecular signal that records the association of independent events.     

Calcium Channel α2δ Subunits: Differential Expression, Function, and Drug Binding

Voltage-activated calcium channels are transmembrane proteins that act as transducers of electrical signals into numerous intracellular activities. On the basis of their electrophysiological properties they are classified as high- and low-voltage-activated calcium channels. High-voltage-activated calcium channels are heterooligomeric proteins consisting of a pore-forming alpha1 subunit and auxiliary alpha2delta, beta, and--in some tissues--gamma subunits. Auxiliary subunits support the membrane trafficking of the alpha1 subunit and modulate the kinetic properties of the channel. In particular, the alpha2delta subunit has been shown to modify the biophysical and pharmacological properties of the alpha1 subunit. The alpha2delta subunit is posttranslationally cleaved to form disulfide-linked alpha2 and, delta proteins, both of which are heavily glycosylated. Recently it was shown that at least four genes encode for alpha2delta subunits which are expressed in a tissue-specific manner. Their biophysical properties were characterized in coexpression studies with high- and low-voltage-activated calcium channels. Mutations in the gene encoding alpha2delta-2 have been found to underlie the ducky phenotype. This mouse mutant is a model for absence epilepsy and is characterized by spike wave seizures and cerebellar ataxia. Alpha2delta subunits can also support pharmacological interactions with drugs that are used for the treatment of epilepsy and neuropathic pain.     

Differential G protein-mediated coupling of D2 dopamine receptors to K+ and Ca2+ currents in rat anterior pituitary cells.

In anterior pituitary cells, dopamine, acting on D2 dopamine receptors, concomitantly reduces calcium currents and increases potassium currents. These dopamine effects require the presence of intracellular GTP and are blocked by pretreatment of the cells with pertussis toxin, suggesting that one or more G protein is involved. To identify the G proteins involved in coupling D2 receptors to these currents, we performed patch-clamp recordings in the whole-cell configuration using pipettes containing affinity-purified polyclonal antibodies raised against either Go alpha, Gi3 alpha, or Gi1,2 alpha. Dialysis with Go alpha antiserum significantly reduced the inhibition of calcium currents induced by dopamine, while increase of potassium currents was markedly attenuated only by Gi3 alpha antiserum. We therefore conclude that in pituitary cells, two different G proteins are involved in the signal transduction mechanism that links D2 receptor activation to a specific modulation of the four types of ionic channels studied here.     

Regulation and function of G alpha protein subunits in Dictyostelium

We have examined the developmental regulation and function of two G alpha protein subunits, G alpha 1 and G alpha 2, from Dictyostelium. G alpha 1 is expressed in vegetative cells through aggregate stages while G alpha 2 is inducible by cAMP pulses and preferentially expressed in aggregation. Our results suggest that G alpha 2 encodes the G alpha protein subunit associated with the cAMP receptor and mediates all known receptor-activated intracellular signal transduction processes, including chemotaxis and gene regulation. G alpha 1 appears to function in both the cell cycle and development. Overexpression of G alpha 1 results in large, multinucleated cells that develop abnormally. The central role that these G alpha proteins play in signal transduction processes and in controlling Dictyostelium development is discussed.     

Calcium channel beta subunit promotes voltage-dependent modulation of alpha 1 B by G beta gamma

Voltage-dependent calcium channels (VDCCs) are heteromultimers composed of a pore-forming alpha1 subunit and auxiliary subunits, including the intracellular beta subunit, which has a strong influence on the channel properties. Voltage-dependent inhibitory modulation of neuronal VDCCs occurs primarily by activation of G-proteins and elevation of the free G beta gamma dimer concentration. Here we have examined the interaction between the regulation of N-type (alpha 1 B) channels by their beta subunits and by G beta gamma dimers, heterologously expressed in COS-7 cells. In contrast to previous studies suggesting antagonism of G protein inhibition by the VDCC beta subunit, we found a significantly larger G beta gamma-dependent inhibition of alpha 1 B channel activation when the VDCC alpha 1 B and beta subunits were coexpressed. In the absence of coexpressed VDCC beta subunit, the G beta gamma dimers, either expressed tonically or elevated via receptor activation, did not produce the expected features of voltage-dependent G protein modulation of N-type channels, including slowed activation and prepulse facilitation, while VDCC beta subunit coexpression restored all of the hallmarks of G beta gamma modulation. These results suggest that the VDCC beta subunit must be present for G beta gamma to induce voltage-dependent modulation of N-type calcium channels.     

Expression and potential functions of G-protein alpha subunits in embryos of Xenopus laevis.

During early embryonic development, many inductive interactions between tissues depend on signal transduction processes. We began to test the possibility that G-proteins participate in the signal transduction pathways that mediate neural induction. The expression during Xenopus development of three G alpha subunits, G alpha 0, G alpha i-1 and G alpha s-1, was characterized. The three maternally expressed genes showed different expression patterns during early development. Whole-mount in situ hybridization revealed that all three genes were expressed almost exclusively in the gastrula ectoderm and predominantly in the neuroectoderm in the neurula embryo. In order to investigate the involvement of these proteins in neural induction, we overexpressed the G-protein alpha subunits by injecting the G alpha mRNAs into fertilized eggs. Overexpression of G alpha s-1 increased the ability of gastrula ectoderm to become induced to neural tissue approximately four-fold. Overexpression of G alpha 0 and G alpha i-1 had less pronounced effects on neural competence, and inhibition of the G alpha 0 and G alpha i-1 proteins by pertussis toxin did not change the neural competence of the exposed gastrula ectoderm. Overexpression of the G alpha 0 and G alpha i-1 genes did, however, inhibit the normal disappearance of the blastocoel during gastrulation, suggesting a role for these G-proteins in regulating this process. The data also suggest a specific role for the G alpha s subunit in mediating the initial phases of neural induction.     

G protein modulation of N-type calcium channels is facilitated by physical interactions between syntaxin 1A and Gbetagamma

The direct modulation of N-type calcium channels by G protein betagamma subunits is considered a key factor in the regulation of neurotransmission. Some of the molecular determinants that govern the binding interaction of N-type channels and Gbetagamma have recently been identified (see, i.e., Zamponi, G. W., Bourinet, E., Nelson, D., Nargeot, J., and Snutch, T. P. (1997) Nature 385, 442-446); however, little is known about cellular mechanisms that modulate this interaction. Here we report that a protein of the presynaptic vesicle release complex, syntaxin 1A, mediates a crucial role in the tonic inhibition of N-type channels by Gbetagamma. When syntaxin 1A was coexpressed with (N-type) alpha(1B) + alpha(2)-delta + beta(1b) channels in tsA-201 cells, the channels underwent a 18 mV negative shift in half-inactivation potential, as well as a pronounced tonic G protein inhibition as assessed by its reversal by strong membrane depolarizations. This tonic inhibition was dramatically attenuated following incubation with botulinum toxin C, indicating that syntaxin 1A expression was indeed responsible for the enhanced G protein modulation. However, when G protein betagamma subunits were concomitantly coexpressed, the toxin became ineffective in removing G protein inhibition, suggesting that syntaxin 1A optimizes, rather than being required for G protein modulation of N-type channels. We also demonstrate that Gbetagamma physically binds to syntaxin 1A, and that syntaxin 1A can simultaneously interact with Gbetagamma and the synprint motif of the N-type channel II-III linker. Taken together, our experiments suggest a mechanism by which syntaxin 1A mediates a colocalization of G protein betagamma subunits and N-type calcium channels, thus resulting in more effective G protein coupling to, and regulation of, the channel. Thus, the interactions between syntaxin, G proteins, and N-type calcium channels are part of the structural specialization of the presynaptic terminal.     

Gi Irks GIRKs.

G protein-activated potassium channels (GIRKs), monitored with the temporal and molecular resolution of electrophysiology, play a key role in the study of signal transduction. GIRKs are activated primarily by the G(beta)(gamma) subunits, but a paper by Peleg et al. (2002 [this issue of Neuron]) demonstrates a role for G(alpha) subunits in suppressing basal activity and supports the idea of a macromolecular complex of G protein, GIRK, and perhaps RGS protein.     

Purification and characterization of Go alpha and three types of Gi alpha after expression in Escherichia coli

Complementary DNAs for the G protein alpha subunits Gi alpha 1, Gi alpha 2, Gi alpha 3, and Go alpha were expressed in Escherichia coli, and the four proteins were purified to homogeneity. The recombinant proteins exchange and hydrolyze guanine nucleotide, are ADP-ribosylated by pertussis toxin, and interact with beta gamma subunits. The rates of dissociation of GDP from Gi alpha 1 and Gi alpha 3 (0.03 min-1) are an order of magnitude slower than that from rGo alpha; release of GDP from Gi alpha 2 is also relatively slow (0.07 min-1). However, the values of kcat for the hydrolysis of GTP by rGo alpha and the three rGi alpha proteins are approximately the same, about 2 min-1 at 20 degrees C. The recombinant proteins restore inhibition of Ca2+ currents in pertussis toxin-treated dorsal root ganglion neurons in response to neuropeptide Y and bradykinin, indicating that the proteins can interact functionally with all necessary components of at least one signal transduction system. The two different receptors function with different arrays of G proteins to mediate their responses, since all four G proteins restored responses to bradykinin, while Gi alpha 2 was inactive with neuropeptide Y. Despite these results, high concentrations of activated Gi alpha proteins are without effect on adenylyl cyclase activity, either in the presence or absence of forskolin or Gs alpha, the G protein that activates adenylyl cyclase. These results are consistent with the hypothesis that G protein beta gamma subunits are primarily responsible for inhibition of adenylyl cyclase activity.     

G protein activation is prerequisite for functional coupling between Galpha/Gbetagamma and tubulin/microtubules

Heterotrimeric G proteins participate in signal transduction by transferring signals from cell surface receptors to intracellular effector molecules. Interestingly, recent results suggest that G proteins also interact with microtubules and participate in cell division and differentiation. It has been shown earlier that both alpha and betagamma subunits of G proteins modulate microtubule assembly in vitro. Since G protein activation and subsequent dissociation of alpha and betagamma subunits are necessary for G proteins to participate in signaling processes, here we asked if similar activation is required for modulation of microtubule assembly by G proteins. We reconstituted Galphabetagamma heterotrimer from myristoylated-Galpha and prenylated-Gbetagamma, and found that the heterotrimer blocks Gi1alpha activation of tubulin GTPase and inhibits the ability of Gbeta1gamma2 to promote in vitro microtubule assembly. Results suggest that G protein activation is required for functional coupling between Galpha/Gbetagamma and tubulin/microtubules, and supports the notion that regulation of microtubules is an integral component of G protein mediated signaling.     

Purification and characterization of a GTP-binding protein serving as pertussis toxin substrate in starfish oocytes

In response to a meiosis-inducing hormone, 1-methyladenine (1-MA), starfish oocytes undergo reinitiation of meiosis with germinal vesicle breakdown. The 1-MA-initiated signal is, however, inhibited by prior microinjection of pertussis toxin into the oocytes (Shilling, F., Chiba, K., Hoshi, M., Kishimoto, T., and Jaffe, L.A. (1989) Dev. Biol. 133, 605-608), suggesting that a pertussis-toxin-sensitive guanine-nucleotide-binding protein (G protein) is involved in the 1-MA-induced signal transduction. Based on these findings, we purified a G protein serving as the substrate of pertussis toxin from the plasma membranes of starfish oocytes. The purified G protein had an alpha beta gamma-trimeric structure consisting of 39-kDa alpha, 37-kDa beta, and 8-kDa gamma subunits. The 39-kDa alpha subunit contained a site for ADP-ribosylation catalyzed by pertussis toxin. The alpha subunit was also recognized by antibodies specific for a common GTP-binding site of many mammalian alpha subunits or a carboxy-terminal ADP-ribosylation site of mammalian inhibitory G-alpha. An antibody raised against mammalian 36-/35-kDa beta subunits strongly reacted with the 37-kDa beta subunit of starfish G protein. The purified starfish G protein had a GTP-binding activity with a high affinity and displayed a low GTPase activity. The activity of the G protein serving as the substrate for pertussis-toxin-catalyzed ADP-ribosylation was inhibited by its association with a non-hydrolyzable GTP analogue. Thus, the starfish G protein appeared to be similar to mammalian G proteins at least in terms of its structure and properties of nucleotide binding and the pertussis toxin substrate. A possible role of the starfish G protein is also discussed in the signal transduction between 1-MA receptors and reinitiation of meiosis with germinal vesicle breakdown.     

Tubulin binds specifically to the signal-transducing proteins, Gs alpha and Gi alpha 1

Participation of cytoskeletal elements in regulation of hormonal response and responsiveness has been suggested by several laboratories. Addition of dimeric tubulin to rat cerebral cortex synaptic membranes causes stable inhibition of adenylyl cyclase, and the molecular basis for this effect appears to require a direct interaction between tubulin and G proteins. To test whether such tubulin-G protein interaction occurred, several purified G proteins were bound to nitrocellulose, and 125I-tubulin overlay studies were performed. 125I-Tubulin bound to the alpha subunits of Gs and Gil with high specificity and an apparent Kd of approximately 130 nM. Other G protein alpha subunits (alpha i2, alpha i3, alpha 0, and transducin) displayed a much lower affinity for tubulin, despite the much closer relationship of those proteins to alpha il than to alpha s. Association of beta gamma subunits with alpha il or alpha s did not alter the binding of tubulin to these G protein heterotrimers, and the binding of a hydrolysis-resistant GTP analog to the alpha subunits was similarly without effect. These results suggest that tubulin forms complexes with specific G proteins and these complexes might provide a locus for the interaction of cytoskeletal components and signal transduction cascades. These results also provide evidence of a functional distinction among the closely related alpha i subtypes.     

Biochemical and genetic analysis of dominant-negative mutations affecting a yeast G-protein gamma subunit.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) consisting of alpha, beta, and gamma subunits mediate signalling between cell surface receptors and intracellular effectors in eukaryotic cells. To define signalling functions of G gamma subunits (STE18 gene product) involved in pheromone response and mating in the yeast Saccharomyces cerevisiae, we isolated and characterized dominant-negative STE18 alleles. We obtained dominant-negative mutations that disrupt C-terminal sequences required for prenylation of G gamma precursors (CAAX box) and that affect residues in the N-terminal half of Ste18p. Overexpression of mutant G gamma subunits in wild-type cells blocked signal transduction; this effect was suppressed upon overexpression of G beta subunits. Mutant G gamma subunits may therefore sequester G beta subunits into nonproductive G beta gamma dimers. Because mutant G gamma subunits blocked the constitutive signal resulting from disruption of the G alpha subunit gene (GPA1), they are defective in functions required for downstream signalling. Ste18p bearing a C107Y substitution in the CAAX box displayed reduced electrophoretic mobility, consistent with a prenylation defect. G gamma subunits carrying N-terminal substitutions had normal electrophoretic mobilities, suggesting that these proteins were prenylated. G gamma subunits bearing substitutions in their N-terminal region or C-terminal CAAX box (C107Y) supported receptor-G protein coupling in vitro, whereas C-terminal truncations caused partial defects in receptor coupling.     

Gβ2 mimics activation kinetic slowing of CaV2.2 channels by noradrenaline in rat sympathetic neurons

Several neurotransmitters and hormones acting through G protein-coupled receptors elicit a voltage-dependent regulation of Ca_V2.2 channels, having profound effects on cell function and the organism. It has been hypothesized that protein–protein interactions define specificity in signal transduction. Yet it is unknown how the molecular interactions in an intracellular signaling cascade determine the specificity of the voltage-dependent regulation induced by a specific neurotransmitter. It has been suspected that specific effector regions on the Gβ subunits of the G proteins are responsible for voltage-dependent regulation. The present study examines whether a neurotransmitter’s specificity can be revealed by simple ion-current kinetic analysis likely resulting from interactions between Gβ subunits and the channel-molecule. Noradrenaline is a neurotransmitter that induces voltage-dependent regulation. By using biochemical and patch-clamp methods in rat sympathetic neurons we examined calcium current modulation induced by each of the five Gβ subunits and found that Gβ₂ mimics activation kinetic slowing of Ca_V2.2 channels by noradrenaline. Furthermore, overexpression of the Gβ₂ isoform reproduces the effect of noradrenaline in the willing–reluctant model. These results advance our understanding on the mechanisms by which signals conveying from a variety of membrane receptors are able to display precise homeostatic responses.     

Molecular cloning of G protein alpha subunits from the central nervous system of the mollusc Lymnaea stagnalis.

The central nervous system of the pond snail, Lymnaea stagnalis, contains many large, identified neurons which can be easily manipulated making it an advantageous model system to elucidate in vivo the architecture of neuronal signal transduction pathways. We have isolated three cDNA clones encoding G protein alpha subunits that are expressed in the Lymnaea CNS, i.e. G alpha o, G alpha s and G alpha i. The deduced proteins exhibit a very high degree of sequence identity to their vertebrate and invertebrate counterparts. The strong conservation of G protein alpha subunits suggests that functional insights into G protein-mediated signalling routes obtained through the experimental amenability of the Lymnaea CNS will have relevance for similar pathways in the mammalian brain.     

Structure and function of voltage-dependent ion channel regulatory beta subunits

Voltage-dependent K(+), Ca(2+), and Na(+) channels play vital roles in basic physiological processes, including management of the action potential, signal transduction, and secretion. They share the common function of passively transporting ions across cell membranes; thus, it would not be surprising if they should exhibit similarities of both structure and mechanism. Indeed, the principal pore-forming (alpha) subunits of each show either exact or approximate 4-fold symmetry and share a similar transmembrane topology, and all are gated by changes in membrane potential. Furthermore, these channels all possess an auxiliary polypeptide, designated the beta subunit, which plays an important role in their regulation. Despite considerable functional semblences and abilities to interact with structurally similar alpha subunits, however, there is considerable structural diversity among the beta subunits. In this review, we discuss the similarities and differences in the structures and functions of the beta subunits of the voltage-dependent K(+), Ca(2+), and Na(+) channels.     

The sigma receptor as a ligand-regulated auxiliary potassium channel subunit

The sigma receptor is a novel protein that mediates the modulation of ion channels by psychotropic drugs through a unique transduction mechanism depending neither on G proteins nor protein phosphorylation. The present study investigated sigma receptor signal transduction by reconstituting responses in Xenopus oocytes. Sigma receptors modulated voltage-gated K+ channels (Kv1.4 or Kv1.5) in different ways in the presence and absence of ligands. Association between Kv1.4 channels and sigma receptors was demonstrated by coimmunoprecipitation. These results indicate a novel mechanism of signal transduction dependent on protein-protein interactions. Domain accessibility experiments suggested a structure for the sigma receptor with two cytoplasmic termini and two membrane-spanning segments. The ligand-independent effects on channels suggest that sigma receptors serve as auxiliary subunits to voltage-gated K+ channels with distinct functional interactions, depending on the presence or absence of ligand.