Melvin I. Simon

DIVERSITY OF G PROTEINS IN SIGNAL TRANSDUCTION

The heterotrimeric guanine nucleotide-binding proteins (G proteins) act as switches that regulate information processing circuits connecting cell surface receptors to a variety of effectors. The G proteins are present in all eukaryotic cells, and they control metabolic, humoral, neural, and developmental functions. More than a hundred different kinds of receptors and many different effectors have been described. The G proteins that coordinate receptor-effector activity are derived from a large gene family. At present, the family is known to contain at least sixteen different genes that encode the alpha subunit of the heterotrimer, four that encode beta subunits, and multiple genes encoding gamma subunits. Specific transient interactions between these components generate the pathways that modulate cellular responses to complex chemical signals.

Slowed recovery of rod photoresponse in mice lacking the GTPase accelerating protein RGS9-1.

Timely deactivation of the α-subunit of the rod G-protein transducin (Gαt) is essential for the temporal resolution of rod vision. Regulators of G-protein signalling (RGS) proteins accelerate hydrolysis of GTP by the α-subunits of heterotrimeric G proteins in vitro. Several retinal RGS proteins can act in vitro as GTPase accelerating proteins (GAP) for Gαt. Recent reconstitution experiments indicate that one of these, RGS9-1, may account for much of the Gαt GAP activity in rod outer segments (ROS). Here we report that ROS membranes from mice lacking RGS9-1 hydrolyse GTP more slowly than ROS membranes from control mice. The Gβ5-L protein that forms a complex with RGS9-1 (ref. 10) was absent from RGS9-/- retinas, although Gβ5-L messenger RNA was still present. The flash responses of RGS9-/- rods rose normally, but recovered much more slowly than normal. We conclude that RGS9-1, probably in a complex with Gβ5-L, is essential for acceleration of hydrolysis of GTP by Gαt and for normal recovery of the photoresponse.

A Diverse Family of GPCRs Expressed in Specific Subsets of Nociceptive Sensory Neurons

In vertebrates, peripheral chemosensory neurons express large families of G protein-coupled receptors (GPCRs), reflecting the diversity and specificity of stimuli they detect. However, somatosensory neurons, which respond to chemical, thermal, or mechanical stimuli, are more broadly tuned. Here we describe a family of approximately 50 GPCRs related to Mas1, called mrgs, a subset of which is expressed in specific subpopulations of sensory neurons that detect painful stimuli. The expression patterns of mrgs thus reveal an unexpected degree of molecular diversity among nociceptive neurons. Some of these receptors can be specifically activated in heterologous cells by RFamide neuropeptides such as NPFF and NPAF, which are analgesic in vivo. Thus, mrgs may regulate nociceptor function and/or development, including the sensation or modulation of pain.

Phosphorylation of three proteins in the signaling pathway of bacterial chemotaxis

Six cytoplasmic che gene products are required for signal transduction in bacterial chemotaxis, but the nature of their biochemical interactions is not known. We show that in vitro the CheA protein becomes autophosphorylated in the presence of ATP. In addition, the phosphate group on CheA can be rapidly transferred to CheB, a protein involved in adaptation to stimuli, or to CheY, a protein involved in the excitation response. The phosphorylation of CheB and CheY is transient; they readily dephosphorylate. We have also found that CheZ, a protein that appears to antagonize CheY function in vivo, accelerates the hydrolysis of the phosphate on CheY. These results suggest that signal transduction in bacterial chemotaxis may involve the flow of phosphate through a cascade of phosphorylated protein intermediates.

Structure of CheA, a Signal-Transducing Histidine Kinase

Histidine kinases allow bacteria, plants, and fungi to sense and respond to their environment. The 2.6 A resolution crystal structure of Thermotoga maritima CheA (290-671) histidine kinase reveals a dimer where the functions of dimerization, ATP binding, and regulation are segregated into domains. The kinase domain is unlike Ser/Thr/Tyr kinases but resembles two ATPases, Gyrase B and Hsp90. Structural analogies within this superfamily suggest that the P1 domain of CheA provides the nucleophilic histidine and activating glutamate for phosphotransfer. The regulatory domain, which binds the homologous receptor-coupling protein CheW, topologically resembles two SH3 domains and provides different protein recognition surfaces at each end. The dimerization domain forms a central four-helix bundle about which the kinase and regulatory domains pivot on conserved hinges to modulate transphosphorylation. Different subunit conformations suggest that relative domain motions link receptor response to kinase activity.

Mechanisms of rhodopsin inactivation in vivo as revealed by a COOH-terminal truncation mutant

Although biochemical experiments suggest that rhodopsin and other receptors coupled to heterotrimeric guanosine triphosphate-binding proteins (G proteins) are inactivated by phosphorylation near the carboxyl (COOH)-terminus and the subsequent binding of a capping protein, little is known about the quenching process in vivo. Flash responses were recorded from rods of transgenic mice in which a fraction of the rhodopsin molecules lacked the COOH-terminal phosphorylation sites. In the single photon regime, abnormally prolonged responses, attributed to activation of individual truncated rhodopsins, occurred interspersed with normal responses. The occurrence of the prolonged responses suggests that phosphorylation is required for normal shutoff. Comparison of normal and prolonged single photon responses indicated that rhodopsin begins to be quenched before the peak of the electrical response and that quenching limits the response amplitude.

Prolonged photoresponses in transgenic mouse rods lacking arrestin

Arrestins are soluble cytoplasmic proteins that bind to G-protein-coupled receptors, thus switching off activation of the G protein and terminating the signalling pathway that triggers the cellular response,. Although visual arrestin has been shown to quench the catalytic activity of photoexcited, phosphorylated rhodopsin in a reconstituted system, its role in the intact rod cell remains unclear because phosphorylation alone reduces the catalytic activity of rhodopsin. Here we have recorded photocurrents of rods from transgenic mice in which one or both copies of the arrestin gene were disrupted. Photoresponses were unaffected when arrestin expression was halved, indicating that arrestin binding is not rate limiting for recovery of the rod photoresponse, as it is in Drosophila,. With arrestin absent, the flash response displayed a rapid partial recovery followed by a prolonged final phase. This behaviour indicates that an arrestin-independent mechanism initiates the quench of rhodopsins catalytic activity and that arrestin completes the quench. The intensity dependence of the photoresponse in rods lacking arrestin further suggests that, although arrestin is required for normal signal termination, it does not participate directly in light adaptation.

TRPV1-expressing primary afferents generate behavioral responses to pruritogens via multiple mechanisms

The mechanisms that generate itch are poorly understood at both the molecular and cellular levels despite its clinical importance. To explore the peripheral neuronal mechanisms underlying itch, we assessed the behavioral responses (scratching) produced by s.c. injection of various pruritogens in PLCβ3- or TRPV1-deficient mice. We provide evidence that at least 3 different molecular pathways contribute to the transduction of itch responses to different pruritogens: 1) histamine requires the function of both PLCβ3 and the TRPV1 channel; 2) serotonin, or a selective agonist, α-methyl-serotonin (α-Me-5-HT), requires the presence of PLCβ3 but not TRPV1, and 3) endothelin-1 (ET-1) does not require either PLCβ3 or TRPV1. To determine whether the activity of these molecules is represented in a particular subpopulation of sensory neurons, we examined the behavioral consequences of selectively eliminating 2 nonoverlapping subsets of nociceptors. The genetic ablation of MrgprD+ neurons that represent ≈90% of cutaneous nonpeptidergic neurons did not affect the scratching responses to a number of pruritogens. In contrast, chemical ablation of the central branch of TRPV1+ nociceptors led to a significant behavioral deficit for pruritogens, including α-Me-5-HT and ET-1, that is, the TRPV1-expressing nociceptor was required, whether or not TRPV1 itself was essential. Thus, TRPV1 neurons are equipped with multiple signaling mechanisms that respond to different pruritogens. Some of these require TRPV1 function; others use alternate signal transduction pathways.

RGS9 modulates dopamine signaling in the basal ganglia.

Regulators of G protein signaling (RGS) modulate heterotrimeric G proteins in part by serving as GTPase-activating proteins for Galpha subunits. We examined a role for RGS9-2, an RGS subtype highly enriched in striatum, in modulating dopamine D2 receptor function. Viral-mediated overexpression of RGS9-2 in rat nucleus accumbens (ventral striatum) reduced locomotor responses to cocaine (an indirect dopamine agonist) and to D2 but not to D1 receptor agonists. Conversely, RGS9 knockout mice showed heightened locomotor and rewarding responses to cocaine and related psychostimulants. In vitro expression of RGS9-2 in Xenopus oocytes accelerated the off-kinetics of D2 receptor-induced GIRK currents, consistent with the in vivo data. Finally, chronic cocaine exposure increased RGS9-2 levels in nucleus accumbens. Together, these data demonstrate a functional interaction between RGS9-2 and D2 receptor signaling and the behavioral actions of psychostimulants and suggest that psychostimulant induction of RGS9-2 represents a compensatory adaptation that diminishes drug responsiveness.

A single lentiviral vector platform for microRNA-based conditional RNA interference and coordinated transgene expression

RNAi is proving to be a powerful experimental tool for the functional annotation of mammalian genomes. The full potential of this technology will be realized through development of approaches permitting regulated manipulation of endogenous gene expression with coordinated reexpression of exogenous transgenes. We describe the development of a lentiviral vector platform, pSLIK (single lentivector for inducible knockdown), which permits tetracycline-regulated expression of microRNA-like short hairpin RNAs from a single viral infection of any naïve cell system. In mouse embryonic fibroblasts, the pSLIK platform was used to conditionally deplete the expression of the heterotrimeric G proteins Gα12 and Gα13 both singly and in combination, demonstrating the Gα13 dependence of serum response element-mediated transcription. In RAW264.7 macrophages, regulated knockdown of Gβ2 correlated with a reduced Ca2+ response to C5a. Insertion of a GFP transgene upstream of the Gβ2 microRNA-like short hairpin RNA allowed concomitant reexpression of a heterologous mRNA during tetracycline-dependent target gene knockdown, significantly enhancing the experimental applicability of the pSLIK system.