Molecular Evolution and Nucleotide Sequences of the Maize Plastid Genes for the α Subunit of Cf1 (atpA) and the Proteolipid Subunit of Cf0 (atpH)

The nucleotide sequences of the maize plastid genes for the alpha subunit of CF1 (atpA) and the proteolipid subunit of CF0 (atpH) are presented. The evolution of these genes among higher plants is characterized by a transition mutation bias of about 2:1 and by rates of synonymous and nonsynonymous substitution which are much lower than similar rates for genes from other sources. This is consistent with the notion that the plastid genome is evolving conservatively in primary sequence. Yet, the mode and tempo of sequence evolution of these and other plastid-encoded coupling factor genes are not the same. In particular, higher rates of nonsynonymous substitution in atpE (the gene for the epsilon subunit of CF1) and higher rates of synonymous substitution in atpH in the dicot vs. monocot lineages of higher plants indicate that these sequences are likely subject to different evolutionary constraints in these two lineages. The 5'- and 3'-transcribed flanking regions of atpA and atpH from maize, wheat and tobacco are conserved in size, but contain few putative regulatory elements which are conserved either in their spatial arrangement or sequence complexity. However, these regions likely contain variable numbers of "species-specific" regulatory elements. The present studies thus suggest that the plastid genome is not a passive participant in an evolutionary process governed by a more rapidly changing, readily adaptive, nuclear compartment, but that novel strategies for the coordinate expression of genes in the plastid genome may arise through rapid evolution of the flanking sequences of these genes.     

MMP-9 Sheds the ��2 Integrin Subunit (CD18) from Macrophages

Activated macrophages are essential effectors of immunity and a rich source of matrix metalloproteinase-9 (MMP-9; gelatinase B). To search for cellular substrates of the enzyme, we subjected wild-type macrophages and macrophages expressing an autoactivating form of pro-MMP-9 (M9A macrophages) to proteomics analysis. Two-dimensional liquid chromatography together with tandem mass spectrometry identified 467 proteins in medium conditioned by M9A and/or wild-type macrophages. Subtractive proteomics identified 18 candidate MMP-9 substrates. Biochemical studies confirmed that two transmembrane proteins, β₂ integrin subunit (CD18) and amyloid protein precursor (APP), were enriched in the medium of M9A macrophages. To identify potential cleavage sites, we synthesized an overlapping library of peptides that spanned 60 residues of the ectodomain and transmembrane domain of β₂ integrin. Active MMP-9 cleaved a single peptide, ECVKGPNVAAIVGGT, at residues corresponding to Ala⁷⁰⁵ and Ile⁷⁰⁶ of the β₂ integrin. Peptides corresponding to this cleavage site were detected by tandem mass spectrometric analysis only in medium from M9A macrophages, strongly supporting the proposal that β₂ integrin is shed by autoactivating MMP-9. Our observations indicate that subtractive proteomics in concert with peptide substrate mapping is a powerful approach for identifying proteolytic substrates and suggest that MMP-9 plays previously unsuspected roles in the regulation and shedding of β₂ integrin.Matrix metalloproteinases (MMPs),¹ a subfamily of metazincins, are a structurally related group of zinc-dependent proteases (1). They are synthesized in latent form as pro-MMPs, and their prodomain must be removed or modified before they are proteolytically active. Some MMPs are secreted, whereas others are anchored to the cell surface, but their proteolytic activity is thought to be confined locally within the secretory pathway at the cell surface and nearby extracellular space (1–3). Individual MMPs have distinct substrate specificities and act on diverse extracellular and membrane proteins, such as chemokines, cell surface adhesion proteins, and extracellular matrix components. Proteolysis by MMPs plays an important role in a wide variety of normal and pathological processes, such as host defense, inflammation, and tumor progression (1–9).High levels of MMP-9 (gelatinase B) are expressed by activated macrophages (10), which are key effector cells of both innate and acquired immunity. In addition to having homeostatic functions, MMP-9 secreted by macrophages has been implicated in aneurysm formation, tumor progression, and disruption of atherosclerotic plaques (8, 9, 11, 12). Although the pathogenesis of those processes is generally thought to involve inappropriate degradation of extracellular matrix proteins, it has become increasingly clear that MMPs cleave a number of diverse substrates to mediate their varied functions (3, 13). Because MMP-9 can accumulate on the cell surface (14), it is likely to act on membrane proteins.To understand the specific roles of individual MMPs in inflammatory and immune responses, it is critical to identify their physiological substrates (3, 15–17). Most studies have focused on identifying substrates by their ability to be cleaved in defined in vitro reactions (18, 19), but this approach is biased in two ways. First, the candidate substrate must be selected a priori. Second, in vitro reactions fail to account for the complexity of the pericellular environment. Another method is to identify sequences in synthetic peptides that MMPs can cleave (20, 21). However, individual MMPs cleave different proteins at a variety of sites rather than at a consensus site. Moreover MMPs often interact with substrates through domains remote from the active site (exosites) (22), and exosites of MMP-2 have been used in a yeast two-hybrid system to trap candidate substrates (23). However, some substrates may bind weakly or not at all to exosites, limiting the utility of this approach for global substrate screening.An emerging strategy for finding MMP substrates is to conduct an unbiased, global search by coupling gel electrophoresis or liquid chromatography with MS-based protein identification. For example, two-dimensional (2D) gel electrophoresis (24) and derivatization of cysteine-containing peptides with an isotope affinity tag (25) have identified candidate substrates for membrane type-1 MMP (MT1-MMP) in plasma and cultured cells. Quantitative approaches using 2D difference gel electrophoresis have identified potential substrates of MMP-2 and MMP-9 in bronchoalveolar lavage fluid (26) and of MMP-9 and the related metalloproteinases ADAM-10 and ADAM-17 in cancer cells (27, 28). Lectin affinity chromatography detected glycosylated proteins that were selectively enriched in medium from a monocyte cell line expressing ADAM-17 and in phorbol ester-stimulated monocytes (16). Recently iTRAQ (isobaric tags for relative and absolute quantitation) labeling was used to identify substrates of MMP-2 (29). It is important to note, however, that proteases can affect protein abundance by pathways not involving proteolysis. Thus, an important limitation of many of these studies is that they fail to provide evidence that proteins with altered abundance in cells expressing a protease are direct substrates for proteolytic cleavage.In the current studies, we used subtractive proteomics to identify proteins enriched in the medium of a macrophage cell line. Subtractive proteomics compares two or more proteomes to identify proteins that are specifically enriched or depleted under certain conditions (30, 31). Our biochemical studies confirmed that two integral membrane proteins, amyloid precursor protein (APP) and the β₂ integrin subunit (CD18), were shed by macrophages expressing autoactivating MMP-9. We next used a peptide substrate mapping strategy to identify potential MMP-9 cleavage sites in β₂ integrin subunit. Targeted MS/MS analysis demonstrated that β₂ integrin subunit peptides with the same cleavage site were detected only in the medium of macrophages expressing autoactivating MMP-9, providing strong evidence that β₂ integrin is a direct substrate for proteolysis. Our observations indicate that subtractive proteomics in concert with peptide substrate mapping is a robust, high throughput technique for identifying cellular substrates that are proteolytically shed from macrophages.     

Gene Splicing of an Invertebrate Beta Subunit (LCavβ) in the N-Terminal and HOOK Domains and Its Regulation of LCav1 and LCav2 Calcium Channels

The accessory beta subunit (Ca_vβ) of calcium channels first appear in the same genome as Ca_v1 L-type calcium channels in single-celled coanoflagellates. The complexity of this relationship expanded in vertebrates to include four different possible Ca_vβ subunits (β₁, β₂, β₃, β₄) which associate with four Ca_v1 channel isoforms (Ca_v1.1 to Ca_v1.4) and three Ca_v2 channel isoforms (Ca_v2.1 to Ca_v2.3). Here we assess the fundamentally-shared features of the Ca_vβ subunit in an invertebrate model (pond snail Lymnaea stagnalis) that bears only three homologous genes: (LCa_v1, LCa_v2, and LCa_vβ). Invertebrate Ca_vβ subunits (in flatworms, snails, squid and honeybees) slow the inactivation kinetics of Ca_v2 channels, and they do so with variable N-termini and lacking the canonical palmitoylation residues of the vertebrate β2a subunit. Alternative splicing of exon 7 of the HOOK domain is a primary determinant of a slow inactivation kinetics imparted by the invertebrate LCa_vβ subunit. LCa_vβ will also slow the inactivation kinetics of LCa_v3 T-type channels, but this is likely not physiologically relevant in vivo. Variable N-termini have little influence on the voltage-dependent inactivation kinetics of differing invertebrate Ca_vβ subunits, but the expression pattern of N-terminal splice isoforms appears to be highly tissue specific. Molluscan LCa_vβ subunits have an N-terminal “A” isoform (coded by exons: 1a and 1b) that structurally resembles the muscle specific variant of vertebrate β1a subunit, and has a broad mRNA expression profile in brain, heart, muscle and glands. A more variable “B” N-terminus (exon 2) in the exon position of mammalian β3 and has a more brain-centric mRNA expression pattern. Lastly, we suggest that the facilitation of closed-state inactivation (e.g. observed in Ca_v2.2 and Ca_vβ₃ subunit combinations) is a specialization in vertebrates, because neither snail subunit (LCa_v2 nor LCa_vβ) appears to be compatible with this observed property.     

Transforming Growth Factor (TGF)-β-activated Kinase 1 (TAK1) Activation Requires Phosphorylation of Serine 412 by Protein Kinase A Catalytic Subunit α (PKACα) and X-linked Protein Kinase (PRKX)

TGF-β-activated kinase 1 (TAK1) is a key kinase in mediating Toll-like receptors (TLRs) and interleukin-1 receptor (IL-1R) signaling. Although TAK1 activation involves the phosphorylation of Thr-184 and Thr-187 residues at the activation loop, the molecular mechanism underlying the complete activation of TAK1 remains elusive. In this work, we show that the Thr-187 phosphorylation of TAK1 is regulated by its C-terminal coiled-coil domain-mediated dimerization in an autophosphorylation manner. Importantly, we find that TAK1 activation in mediating downstream signaling requires an additional phosphorylation at Ser-412, which is critical for TAK1 response to proinflammatory stimuli, such as TNF-α, LPS, and IL-1β. In vitro kinase and shRNA-based knockdown assays reveal that TAK1 Ser-412 phosphorylation is regulated by cAMP-dependent protein kinase catalytic subunit α (PKACα) and X-linked protein kinase (PRKX), which is essential for proper signaling and proinflammatory cytokine induction by TLR/IL-1R activation. Morpholino-based in vivo knockdown and rescue studies show that the corresponding site Ser-391 in zebrafish TAK1 plays a conserved role in NF-κB activation. Collectively, our data unravel a previously unknown mechanism involving TAK1 phosphorylation mediated by PKACα and PRKX that contributes to innate immune signaling.     

Selection and Characterization of Artificial Proteins Targeting the Tubulin α Subunit

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Subunit structure and gene control of mouse NADP—malate dehydrogenase

The NADP-dependent malate dehydrogenase (MDH) in the supernatant fraction of mouse tissues is known to occur in two allelic forms which are electrophoretically distinguishable; each produces a single band in starch gel. We have investigated the subunit structure and synthesis of NADP—MDH through electrophoretic patterns obtained from several experimental sources. (1) Heterozygotes containing both alleles yield a five-banded pattern. The bands are in an approximate frequency of 1:4:6:4:1; the two extremes correspond to the pure types and the three intermediates are presumably hybrid enzymes. The NADP—MDH molecule therefore appears to be a tetramer. (2) In muscle heterokaryons of allophenic mice (with homozygous nuclei of each genotype within a common cytoplasm), hybrid enzymes are formed; they are not formed in other allophenic tissues. Therefore the gene at this locus codes only for monomeric subunits and the tetramer is assembled in a second step in the cytoplasm. Also, both genes must function in a nucleus when the locus is active (e.g., in F₁ uninucleated cells). (3) Dissociation in vitro of mixtures of both pure types of enzymes, followed by reassociation among fragments, leads to a three-banded pattern, even after repeated cycles. Thus the tetramer must cleave in a fixed plane, to form dimers, which reassociate, rather than in a random fashion to form monomers. The most likely interpretation is that mouse NADP—MDH is an example of the type of tetramer postulated by Monod et al. (1965) and termed isologous. The dimers are held symmetrically in the tetrameric conformation by relatively weak forces; the monomeric subunits comprising the dimer are held together by stronger forces.These investigations were supported by U.S.P.H.S. grants No. HD-01646, CA-06927, and FR-05539, and by an appropriation from the Commonwealth of Pennsylvania.     

Influence of GABA(A) Receptor α Subunit Isoforms on the Benzodiazepine Binding Site

Classical benzodiazepines, such as diazepam, interact with α_xβ₂γ₂ GABA_A receptors, x = 1, 2, 3, 5 and modulate their function. Modulation of different receptor isoforms probably results in selective behavioural effects as sedation and anxiolysis. Knowledge of differences in the structure of the binding pocket in different receptor isoforms is of interest for the generation of isoform-specific ligands. We studied here the interaction of the covalently reacting diazepam analogue 3-NCS with α₁S204Cβ₂γ₂, α₁S205Cβ₂γ₂ and α₁T206Cβ₂γ₂ and with receptors containing the homologous mutations in α₂β₂γ₂, α₃β₂γ₂, α₅β_1/2γ₂ and α₆β₂γ₂. The interaction was studied using radioactive ligand binding and at the functional level using electrophysiological techniques. Both strategies gave overlapping results. Our data allow conclusions about the relative apposition of α₁S204Cβ₂γ₂, α₁S205Cβ₂γ₂ and α₁T206Cβ₂γ₂ and homologous positions in α₂, α₃, α₅ and α₆ with C-atom adjacent to the keto-group in diazepam. Together with similar data on the C-atom carrying Cl in diazepam, they indicate that the architecture of the binding site for benzodiazepines differs in each GABA_A receptor isoform α₁β₂γ₂, α₂β₂γ₂, α₃β₂γ₂, α₅β_1/2γ₂ and α₆β₂γ₂.     

Synthesis and characterization of tris(β-diketonato)technetium(III) and -(IV) complexes

The preparations and properties of tris(dipivaloylmethanato)technetium(III), tris(trifluoroacetylacetonato)technetium(III), and tris(hexafluoroacetonato)technetium(III) are described. The oxidation of the dipivaloyl derivative to tris(dipivaloyl)technetium(IV) hexafluorophosphate was shown to take place readily. Voltammetric studies and magnetic resonance results on the new complexes are reported. The large shifts observed for the complexes seem to be due to a contact interaction.     

Modulation of NMDA Receptor Subunit mRNA in Butorphanol-Tolerant and —Withdrawing Rats

The NMDA receptor has been implicated in opioid tolerance and withdrawal. The effects of continuous infusion of butorphanol on the modulation of NMDA receptor subunit NR1, NR2A, NR2B, and NR2C gene expression were investigated by using in situ hybridization technique. Continuous intracerebroventricular (i.c.v.) infusion with butorphanol (26 nmol/l/h) resulted in significant modulations in the NR1, NR2A, and NR2B mRNA levels. The level of NR1 mRNA was significantly decreased in the cerebral cortex, thalamus, and CA1 area of hippocampus in butorphanol tolerant and withdrawal (7 h after stopping the infusion) rats. The NR2A mRNA was significantly decreased in the CA1 and CA3 of hippocampus in tolerant rats and increased in the cerebral cortex and dentate gyrus in butorphanol withdrawal rats. NR2B subunit mRNA was decreased in the cerebral cortex, caudate putamen, thalamus, CA3 of hippocampus in butorphanol withdrawal rats. No changes of NR1, NR2A, NR2C subunit mRNA in the cerebellar granule cell layer were observed in either butorphanol tolerant or withdrawal rats. Using quantitative ligand autoradiography, the binding of NMDA receptor ligand [³H]MK-801 was increased significantly in all brain regions except in the thalamus and hippocampus, at the 7 hr after stopping the butorphanol infusion. These results suggest that region-specific changes of NMDA receptor subunit mRNA (NR 1 and NR2) as well as NMDA receptor binding ([³H]MK-801) are involved in the development of tolerance to and withdrawal from butorphanol.     

Glucosidase II β Subunit Modulates N-Glycan Trimming in Fission Yeasts and Mammals

Glucosidase II (GII) plays a key role in glycoprotein biogenesis in the endoplasmic reticulum (ER). It is responsible for the sequential removal of the two innermost glucose residues from the glycan (Glc₃Man₉GlcNAc₂) transferred to Asn residues in proteins. GII participates in the calnexin/calreticulin cycle; it removes the single glucose unit added to folding intermediates and misfolded glycoproteins by the UDP-Glc:glycoprotein glucosyltransferase. GII is a heterodimer whose α subunit (GIIα) bears the glycosyl hydrolase active site, whereas its β subunit (GIIβ) role is controversial and has been reported to be involved in GIIα ER retention and folding. Here, we report that in the absence of GIIβ, the catalytic subunit GIIα of the fission yeast Schizosaccharomyces pombe (an organism displaying a glycoprotein folding quality control mechanism similar to that occurring in mammalian cells) folds to an active conformation able to hydrolyze p-nitrophenyl α-d-glucopyranoside. However, the heterodimer is required to efficiently deglucosylate the physiological substrates Glc₂Man₉GlcNAc₂ (G2M9) and Glc₁Man₉GlcNAc₂ (G1M9). The interaction of the mannose 6-phosphate receptor homologous domain present in GIIβ and mannoses in the B and/or C arms of the glycans mediates glycan hydrolysis enhancement. We present evidence that also in mammalian cells GIIβ modulates G2M9 and G1M9 trimming.     

Mapping of the Mouse Actin Capping Protein α Subunit Genes and Pseudogenes

Capping protein (CP), a heterodimer of α and β subunits, is found in all eukaryotes. CP binds to the barbed ends of actin filamentsin vitroand controls actin assembly and cell motilityin vivo.Vertebrates have three α isoforms (α1, α2, α3) produced from different genes, whereas lower organisms have only one gene and one isoform. We isolated genomic clones corresponding to the α subunits of mouse CP and found three α1 genes, two of which are pseudogenes, and a single gene for both α2 and α3. Their chromosomal locations were identified by interspecies backcross mapping. The α1 gene (Cappa1) mapped to Chromosome 3 betweenD3Mit11andD3Mit13.The α1 pseudogenes (Cappa1-ps1andCappa1-ps2) mapped to Chromosomes 1 and 9, respectively. The α2 gene (Cappa2) mapped to Chromosome 6 nearPtn.The α3 gene (Cappa3) also mapped to Chromosome 6, approximately 68 cM distal fromCappa2nearKras2.One mouse mutation,de,maps in the vicinity of the α1 gene. No known mouse mutations map to regions near the α2 or α3 genes.     

Syntheses of (±)-Epoformin (Desoxyepoxydon) and (±)-Epiepoformin (Desoxyepiepoxydon)

Boron is an essential nutrient for plants, but it is toxic in excess. Transgenic rice plants expressing an Arabidopsis thaliana borate efflux transporter gene, AtBOR4, at a low level exhibited increased tolerance to excess boron. Those lines with high levels of expression exhibited reduced growth. These findings suggest a potential of the borate transporter BOR4 for the generation of high-boron tolerant rice.     

Large Conductance Voltage- and Ca2+-gated Potassium (BK) Channel β4 Subunit Influences Sensitivity and Tolerance to Alcohol by Altering Its Response to Kinases

Tolerance is a well described component of alcohol abuse and addiction. The large conductance voltage- and Ca²⁺-gated potassium channel (BK) has been very useful for studying molecular tolerance. The influence of association with the β4 subunit can be observed at the level of individual channels, action potentials in brain slices, and finally, drinking behavior in the mouse. Previously, we showed that 50 mm alcohol increases both α and αβ4 BK channel open probability, but only α BK develops acute tolerance to this effect. Currently, we explore the possibility that the influence of the β4 subunit on tolerance may result from a striking effect of β4 on kinase modulation of the BK channel. We examine the influence of the β4 subunit on PKA, CaMKII, and phosphatase modulation of channel activity, and on molecular tolerance to alcohol. We record from human BK channels heterologously expressed in HEK 293 cells composed of its core subunit, α alone (Insertless), or co-expressed with the β4 BK auxiliary subunit, as well as, acutely dissociated nucleus accumbens neurons using the cell-attached patch clamp configuration. Our results indicate that BK channels are strongly modulated by activation of specific kinases (PKA and CaMKII) and phosphatases. The presence of the β4 subunit greatly influences this modulation, allowing a variety of outcomes for BK channel activity in response to acute alcohol.     

Synthesis of (±)-Homosarkomycin and (±)-Rosaprostol

(±)-Homosarkomycin (2) and (±)-rosaprostol (3) were synthesized from (±)-methyl 2-oxo-bicyclo[3.1.0]hexane-1-carboxylate (1) by using the nucleophilic ring opening reaction on the double-activated cyclopropane ring as the key step.     

Synthesis of (±)-Lamprolobine and (±)-Epilamprolobine

(±)-Lamprolobine, the (+)-enatiomer of which was isolated from the leaves of Lamprolobium fruticosum, and (±)-epilamprolobine were synthesized from δ-valerolactam.     

Functional Characterization of Wiskott-Aldrich Syndrome Protein and Scar Homolog (WASH), a Bi-modular Nucleation-promoting Factor Able to Interact with Biogenesis of Lysosome-related Organelle Subunit 2 (BLOS2) and γ-Tubulin

The Arp2/3 complex is essential for actin filament nucleation in a variety of cellular processes. The activation of the Arp2/3 complex is mediated by nucleation-promoting factors, such as the Wiskott-Aldrich syndrome family proteins, which share a WCA (WH2 domain, central region, acidic region) catalytic module at the C-terminal region, required for Arp2/3 activation, but diverge at the N-terminal region, required for binding to specific activators. Here, we report the characterization of WASH, a new member of the WAS family that has nucleation-promoting factor activity and recently has been demonstrated to play a role in endosomal sorting. We found that overexpression of the WASH-WCA domain induced disruption of the actin cytoskeleton, whereas overexpression of full-length WASH in mammalian cells did not affect stress fiber organization. Furthermore, our analysis has revealed that nerve growth factor treatment of PC12 cells overexpressing full-length WASH leads to disruption of the actin cytoskeleton. We have also found that WASH interacts through its N-terminal region with BLOS2, a centrosomal protein belonging to the BLOC-1 complex that functions as a scaffolding factor in the biogenesis of lysosome-related organelles. In addition to BLOS2, WASH also interacts with centrosomal γ-tubulin and with pallidin, an additional component of the BLOC-1 complex. Collectively, our data propose that WASH is a bimodular protein in which the C terminus is involved in Arp2/3-mediated actin nucleation, whereas the N-terminal portion is required for its regulation and localization in the cells. Moreover, our data suggest that WASH is also a component of the BLOC-1 complex that is associated with the centrosomes.     

Structural Determinants of G-protein �� Subunit Selectivity by Regulator of G-protein Signaling 2 (RGS2)

“Regulator of G-protein signaling” (RGS) proteins facilitate the termination of G protein-coupled receptor (GPCR) signaling via their ability to increase the intrinsic GTP hydrolysis rate of Gα subunits (known as GTPase-accelerating protein or “GAP” activity). RGS2 is unique in its in vitro potency and selectivity as a GAP for Gα_q subunits. As many vasoconstrictive hormones signal via G_q heterotrimer-coupled receptors, it is perhaps not surprising that RGS2-deficient mice exhibit constitutive hypertension. However, to date the particular structural features within RGS2 determining its selectivity for Gα_q over Gα_i/o substrates have not been completely characterized. Here, we examine a trio of point mutations to RGS2 that elicits Gα_i-directed binding and GAP activities without perturbing its association with Gα_q. Using x-ray crystallography, we determined a model of the triple mutant RGS2 in complex with a transition state mimetic form of Gα_i at 2.8-Å resolution. Structural comparison with unliganded, wild type RGS2 and of other RGS domain/Gα complexes highlighted the roles of these residues in wild type RGS2 that weaken Gα_i subunit association. Moreover, these three amino acids are seen to be evolutionarily conserved among organisms with modern cardiovascular systems, suggesting that RGS2 arose from the R4-subfamily of RGS proteins to have specialized activity as a potent and selective Gα_q GAP that modulates cardiovascular function.G protein-coupled receptors (GPCRs)⁴ form an interface between extracellular and intracellular physiology, as they convert hormonal signals into changes in intracellular metabolism and ultimately cell phenotype and function (1–3). GPCRs are coupled to their underlying second messenger systems by heterotrimeric guanine nucleotide-binding protein (“G-proteins”) composed of three subunits: Gα, Gβ, and Gγ. Four general classes of Gα subunits have been defined based on functional couplings (in the GTP-bound state) to various effector proteins. G_s subfamily Gα subunits are stimulatory to membrane-bound adenylyl cyclases that generate the second messenger 3′,5′-cyclic adenosine monophosphate (cAMP); conversely, G_i subfamily Gα subunits are generally inhibitory to adenylyl cyclases (4). G_12/13 subfamily Gα subunits activate the small G-protein RhoA through stimulation of the GEF subfamily of RGS proteins, namely p115-RhoGEF, LARG, and PDZ-RhoGEF (5). G_q subfamily Gα subunits are potent activators of phospholipase-Cβ enzymes that generate the second messengers diacylglycerol and inositol triphosphate (6); more recently, two additional Gα_q effector proteins have been described: the receptor kinase GRK2 and the RhoA nucleotide exchange factor p63RhoGEF (7, 8).The duration of GPCR signaling is controlled by the time Gα remains bound to GTP before its hydrolysis to GDP. RGS proteins are key modulators of GPCR signaling by virtue of their ability to accelerate the intrinsic GTP hydrolysis activity of Gα subunits (reviewed in Refs. 9 and 10). RGS2/G0S8, one of the first mammalian RGS proteins identified (11) and member of the R4-subfamily (10), has a critical role in the maintenance of normostatic blood pressure both in mouse models (12, 13) and in humans (14, 15); additionally, Rgs2-deficient mice exhibit impaired aggression and increased anxiety (16, 17), behavioral phenotypes with potential human clinical correlates (18, 19).Although many RGS proteins are promiscuous and thus act on multiple Gα substrates in vitro (e.g. Ref. 20), RGS2 exhibits exquisite specificity for Gα_q in biochemical binding assays and single turnover GTPase acceleration assays (20, 21). Consistent with this in vitro selectivity,⁵ mice deficient in RGS2 uniquely exhibit constitutive hypertension and prolonged responses to vasoconstrictors, as would be expected upon loss of a potent negative regulator of Gα_q that mediates signaling from various vasoconstrictive hormones such as angiotensin II, endothelin, thrombin, norepinephrine, and vasopressin (22). In addition, RGS2-deficient mice respond to sustained pressure overload with an accelerated time course of maladaptive cardiac remodeling (23), a pathophysiological response that evokes myocardial hypertrophy known to be critically dependent on Gα_q signaling (24, 25).To gain insight into the structural basis of the unique Gα substrate selectivity exhibited by RGS2, a series of point mutants in RGS2 were evaluated that enable this protein to bind and accelerate GTP hydrolysis by Gα_i; we subsequently delineated the structural determinants of the Gα_i/mutant RGS2 interaction using x-ray crystallography. Three key positions, first identified by Heximer and colleagues (21) and highlighted in our structural studies as key determinants of RGS2 substrate selection, were also found to be conserved throughout the evolution of the RGS2 protein in a manner suggestive of specialization toward cardiovascular signaling modulation.