David M. Berman

Structure of RGS4 Bound to AlF4−-Activated Giα1: Stabilization of the Transition State for GTP Hydrolysis

RGS proteins are GTPase activators for heterotrimeric G proteins. We report here the 2.8 A resolution crystal structure of the RGS protein RGS4 complexed with G(i alpha1)-Mg2+-GDP-AlF4 . Only the core domain of RGS4 is visible in the crystal. The core domain binds to the three switch regions of G(i alpha1), but does not contribute catalytic residues that directly interact with either GDP or AlF4-. Therefore, RGS4 appears to catalyze rapid hydrolysis of GTP primarily by stabilizing the switch regions of G(i alpha1), although the conserved Asn-128 from RGS4 could also play a catalytic role by interacting with the hydrolytic water molecule or the side chain of Gln-204. The binding site for RGS4 on G(i alpha1) is also consistent with the activity of RGS proteins as antagonists of G(alpha) effectors.

GAIP and RGS4 Are GTPase-Activating Proteins for the Gi Subfamily of G Protein α Subunits

A novel class of regulators of G protein signaling (RGS) proteins has been identified recently. Genetic evidence suggests that RGS proteins inhibit G protein-mediated signaling at the level of the receptor-G protein interaction or the G protein alpha subunit itself. We have found that two RGS family members, GAIP and RGS4, are GTPase-activating proteins (GAPs), accelerating the rate of GTP hydrolysis by Gi alpha 1 at least 40-fold. All Gi subfamily members assayed were substrates for these GAPs; Gs alpha was not. RGS4 activates the GTPase activity of certain Gi alpha 1 mutants (e.g., R178C), but not others (e.g., Q204L). The GAP activity of RGS proteins is consistent with their proposed role as negative regulators of G protein-mediated signaling.

Mammalian RGS Proteins: Barbarians at the Gate

Hundreds or thousands of chemical and physical stimuli regulate the functions of eukaryotic cells by controlling the activities of a surprisingly small number of core signaling units that have been duplicated and adapted to achieve the necessary diversity. The most prevalent of these units, at least in animal cells, are three-protein modules consisting of signal recognition elements (receptors) and signal generators (effectors) whose activities are linked and coordinated by heterotrimeric guanine nucleotide-binding proteins or G proteins. Collectively, mammalian cells contain hundreds of G proteincoupled receptors and dozens of effectors. It is difficult to count functionally distinct G proteins because we do not understand the significance of the heterogeneity offered by the possible combination of 16 a, 5 b, and at least 12 g subunits (for reviews, see Refs. 1–5). GDP-bound G protein a subunits have high affinity for a tight complex of b and g subunits. This interaction of a with bg occludes the sites of interaction of both of these signaling molecules with downstream effectors, and the inactive state is maintained by an extremely slow rate of dissociation of GDP from the oligomer (k ; 0.01/min). An agonist-bound receptor (typically a 35–60-kDa protein with seven plasma membranespanning helices) activates an appropriate G protein by poorly understood interactions that promote dissociation of GDP. High intracellular concentrations of GTP ensure a transient existence of the nucleotide-free G protein, and binding of GTP causes conformational changes in a that result in dissociation of GTP-a from bg. Both of these complexes can then activate or inhibit signaling pathways by engaging in interactions with effectors such as adenylyl cyclases, phospholipases, cyclic nucleotide phosphodiesterases, and ion channels. Termination of signaling is dependent on the GTPase activity of a. Typically slow (kcat ; 4/min) hydrolysis of GTP to GDP (which remains protein bound) promotes dissociation of a from effectors and reassociation with bg. The slow intrinsic rate of GTP hydrolysis by Ga proteins is regulated by interactions with so-called GTPase-activating proteins or GAPs. GAPs were first recognized as regulators of protein synthesis factors and low molecular weight GTPases such as Ras. It is now appreciated that certain effectors in G protein-regulated pathways act as GAPs on cognate Ga proteins (6, 7) and that there exists a large, newly discovered family of GAPs for Ga proteins known as regulators of G protein signaling or RGS proteins. Although one critical biochemical property of this novel RGS protein family has been defined, knowledge of the requisite regulation of these regulators is negligible. There are hints, however, that these proteins may be poised at centers of signaling to intercept activated G proteins, acting, from a G protein’s point of view, as “barbarians at the gate” of cellular signaling.

THE GTPASE-ACTIVATING PROTEIN RGS4 STABILIZES THE TRANSITION STATE FOR NUCLEOTIDE HYDROLYSIS

RGS proteins constitute a newly appreciated group of negative regulators of G protein signaling. Discovered by genetic screens in yeast, worms, and other organisms, two mammalian RGS proteins, RGS4 and GAIP, act as GTPase-activating proteins for members of the Gi family of G protein α subunits. We have purified recombinant RGS4 to homogeneity and demonstrate that it acts catalytically to stimulate GTP hydrolysis by Gi proteins. Furthermore, RGS4 stabilizes the transition state for GTP hydrolysis, as evidenced by its high affinity for the GDP-AlF4−-bound forms of Goα and Giα and its relatively low affinity for the GTPγS- and GDP-bound forms of these proteins. Consequently, RGS4 is most likely not a downstream effector for activated Gα subunits. All members of the Gi subfamily of proteins tested are substrates for RGS4 (including Gtα and Gzα); the protein has lower affinity for Gqα, and it does not stimulate the GTPase activity of Gsα or G12α.

RGS Proteins Determine Signaling Specificity of Gq-coupled Receptors

Regulators of G protein signaling (RGS) proteins accelerate GTP hydrolysis by Gα subunits, thereby attenuating signaling. RGS4 is a GTPase-activating protein for Giand Gq class α subunits. In the present study, we used knockouts of Gq class genes in mice to evaluate the potency and selectivity of RGS4 in modulating Ca2+ signaling transduced by different Gq-coupled receptors. RGS4 inhibited phospholipase C activity and Ca2+ signaling in a receptor-selective manner in both permeabilized cells and cells dialyzed with RGS4 through a patch pipette. Receptor-dependent inhibition of Ca2+ signaling by RGS4 was observed in acini prepared from the rat and mouse pancreas. The response of mouse pancreatic acini to carbachol was about 4- and 33-fold more sensitive to RGS4 than that of bombesin and cholecystokinin (CCK), respectively. RGS1 and RGS16 were also potent inhibitors of Gq-dependent Ca2+signaling and acted in a receptor-selective manner. RGS1 showed approximately 1000-fold higher potency in inhibiting carbachol than CCK-dependent signaling. RGS16 was as effective as RGS1 in inhibiting carbachol-dependent signaling but only partially inhibited the response to CCK. By contrast, RGS2 inhibited the response to carbachol and CCK with equal potency. The same pattern of receptor-selective inhibition by RGS4 was observed in acinar cells from wild type and several single and double Gq class knockout mice. Thus, these receptors appear to couple Gq class α subunit isotypes equally. Difference in receptor selectivity of RGS proteins action indicates that regulatory specificity is conferred by interaction of RGS proteins with receptor complexes.

Characterization and chromosomal mapping of a human steroid 5α-reductase gene and pseudogene and mapping of the mouse homologue ☆

The enzyme steroid 5 alpha-reductase catalyzes the conversion of testosterone into the more powerful androgen, dihydrotestosterone. We previously described the cloning of rat and human cDNAs that encode steroid 5 alpha-reductase and their expression in oocytes and cultured cells. Here, we report the isolation, characterization, and chromosomal mapping of two human steroid 5 alpha-reductase genes. One gene (symbol SRD5A1) is functional, contains five exons separated by four introns, and maps to the distal short arm of chromosome 5. Two informative restriction fragment length polymorphisms are present in exons 1 and 2 of this gene. A second gene (symbol SRD5AP1) has all of the hallmarks of a processed pseudogene and was mapped to the q24-qter region of the X chromosome. In the mouse, a single steroid 5 alpha-reductase gene (Srd5 alpha-1) is linked to Xmv-13 on chromosome 13.

The Molecular Genetics of Steroid 5α-Reductases

Publisher Summary This chapter discusses the molecular genetics of steroid 5α-reductases. 5α-reductase plays a central role in androgen action by catalyzing the conversion of testosterone into the more potent hormone dihydrotestosterone. Like other steroid and thyroid hormones, testosterone and dihydrotestosterone activate responsive genes by binding to the androgen receptor—a member of the steroid hormone receptor family of transcriptional activator proteins. Despite this mechanistic similarity, androgen action differs from other steroid hormones in that two different steroids interact with the same receptor to bring about different physiological effects. Testosterone bound to the androgen receptor is responsible for the regulation of gonadotropin production, spermatogenesis, and the formation of the internal male genitalia—epididymis, seminal vesicles, and vas deferens—from wolffian duct anlagen during phenotypic sexual differentiation in the male embryo. In contrast, dihydrotestosterone bound to the same androgen receptor brings about the formation of the male external genitalia—penis and scrotum—and prostate from urogenital sinus primordium in the male embryo and is required for sexual maturation at puberty.