Cloning and mitochondrial localization of full-length D-AKAP2, a protein kinase A anchoring protein.

Differential compartmentalization of signaling molecules in cells and tissues is being recognized as an important mechanism for regulating the specificity of signal transduction pathways. A kinase anchoring proteins (AKAPs) direct the subcellular localization of protein kinase A (PKA) by binding to its regulatory (R) subunits. Dual specific AKAPs (D-AKAPs) interact with both RI and RII. A 372-residue fragment of mouse D-AKAP2 with a 40-residue C-terminal PKA binding region and a putative regulator of G protein signaling (RGS) domain was previously identified by means of a yeast two-hybrid screen. Here, we report the cloning of full-length human D-AKAP2 (662 residues) with an additional putative RGS domain, and the corresponding mouse protein less the first two exons (617 residues). Expression of D-AKAP2 was characterized by using mouse tissue extracts. Full-length D-AKAP2 from various tissues shows different molecular weights, possibly because of alternative splicing or posttranslational modifications. The cloned human gene product has a molecular weight similar to one of the prominent mouse proteins. In vivo association of D-AKAP2 with PKA in mouse brain was demonstrated by using cAMP agarose pull-down assay. Subcellular localization for endogenous mouse, rat, and human D-AKAP2 was determined by immunocytochemistry, immunohistochemistry, and tissue fractionation. D-AKAP2 from all three species is highly enriched in mitochondria. The mitochondrial localization and the presence of RGS domains in D-AKAP2 may have important implications for its function in PKA and G protein signal transduction.

A G protein gamma subunit-like domain shared between RGS11 and other RGS proteins specifies binding to Gbeta5 subunits.

Regulators of G protein signaling (RGS) proteins act as GTPase-activating proteins (GAPs) toward the alpha subunits of heterotrimeric, signal-transducing G proteins. RGS11 contains a G protein gamma subunit-like (GGL) domain between its Dishevelled/Egl-10/Pleckstrin and RGS domains. GGL domains are also found in RGS6, RGS7, RGS9, and the Caenorhabditis elegans protein EGL-10. Coexpression of RGS11 with different Gbeta subunits reveals specific interaction between RGS11 and Gbeta5. The expression of mRNA for RGS11 and Gbeta5 in human tissues overlaps. The Gbeta5/RGS11 heterodimer acts as a GAP on Galphao, apparently selectively. RGS proteins that contain GGL domains appear to act as GAPs for Galpha proteins and form complexes with specific Gbeta subunits, adding to the combinatorial complexity of G protein-mediated signaling pathways.

Dynamic regulation of RGS2 suggests a novel mechanism in G-protein signaling and neuronal plasticity.

Long-term neuronal plasticity is known to be dependent on rapid de novo synthesis of mRNA and protein, and recent studies provide insight into the molecules involved in this response. Here, we demonstrate that mRNA encoding a member of the regulator of G-protein signaling (RGS) family, RGS2, is rapidly induced in neurons of the hippocampus, cortex, and striatum in response to stimuli that evoke plasticity. Although several members of the RGS family are expressed in brain with discrete neuronal localizations, RGS2 appears unique in that its expression is dynamically responsive to neuronal activity. In biochemical assays, RGS2 stimulates the GTPase activity of the alpha subunit of Gq and Gi1. The effect on Gi1 was observed only after reconstitution of the protein in phospholipid vesicles containing M2 muscarinic acetylcholine receptors. RGS2 also inhibits both Gq- and Gi-dependent responses in transfected cells. These studies suggest a novel mechanism linking neuronal activity and signal transduction.

GTPase activating specificity of RGS12 and binding specificity of an alternatively spliced PDZ (PSD-95/Dlg/ZO-1) domain.

Regulator of G-protein signaling (RGS) proteins increase the intrinsic guanosine triphosphatase (GTPase) activity of G-protein alpha subunits in vitro, but how specific G-protein-coupled receptor systems are targeted for down-regulation by RGS proteins remains uncharacterized. Here, we describe the GTPase specificity of RGS12 and identify four alternatively spliced forms of human RGS12 mRNA. Two RGS12 isoforms of 6.3 and 5.7 kilobases (kb), encoding both an N-terminal PDZ (PSD-95/Dlg/ZO-1) domain and the RGS domain, are expressed in most tissues, with highest levels observed in testis, ovary, spleen, cerebellum, and caudate nucleus. The 5.7-kb isoform has an alternative 3' end encoding a putative C-terminal PDZ domain docking site. Two smaller isoforms, of 3.1 and 3.7 kb, which lack the PDZ domain and encode the RGS domain with and without the alternative 3' end, respectively, are most abundantly expressed in brain, kidney, thymus, and prostate. In vitro biochemical assays indicate that RGS12 is a GTPase-activating protein for Gi class alpha subunits. Biochemical and interaction trap experiments suggest that the RGS12 N terminus acts as a classical PDZ domain, binding selectively to C-terminal (A/S)-T-X-(L/V) motifs as found within both the interleukin-8 receptor B (CXCR2) and the alternative 3' exon form of RGS12. The presence of an alternatively spliced PDZ domain within RGS12 suggests a mechanism by which RGS proteins may target specific G-protein-coupled receptor systems for desensitization.

Evidence for the shuttle model for Gs alpha activation of adenylyl cyclase.

Knowledge of the nature of the interaction between the stimulatory G protein (Gs) and the adenylyl cyclase catalytic unit (C) is essential for interpreting the effects of Gs mutations and expression levels on cellular response to a wide variety of hormones, drugs, and neurotransmitters. It has been proposed that beta-adrenergic receptor activation of adenylyl cyclase occurs either by a two-step "shuttle" mechanism where the receptor activates Gs independently of cyclase followed by Gs alpha activation of cyclase independent of the receptor; or the receptor activates a "precoupled" Gs-C complex in a single step. Simulations of the two models revealed that the two forms of activation are distinguishable by the effect of Gs levels on epinephrine-stimulated EC50 values for cyclase activation; specifically, the shuttle model predicts an increased potency of epinephrine stimulation as levels of Gs alpha increase. To address this problem, S49 cyc- cells were stably transfected with the gene for Gs alpha(long) regulated by the MMTV LTR promoter, which allowed for an induction of Gs alpha(long) expression levels over a 40-fold range by incubation of the cells for various times with 5 microM dexamethasone. Expression of Gs alpha was strongly correlated to the appearance of GTP shifts in the competitive binding of epinephrine with [125I]iodocyanopindolol to the beta-adrenergic receptors and epinephrine-stimulated adenylyl cyclase activity. Most importantly, high expression of Gs alpha resulted in lower EC50 values for epinephrine and prostaglandin E1 stimulation of adenylyl cyclase activity. The decrease in EC50 did not occur as a result of a change in beta2-adrenergic receptor, Gi alpha, G betagamma, or adenylyl cyclase levels. These novel findings demonstrate that a change in the level of a protein downstream of a plasma membrane receptor can influence hormone potency. We explain these results by using kinetic arguments to suggest that some fraction of hormone-activated adenylyl cyclase occurs via a shuttle mechanism, and not a purely precoupled mechanism.

Examination of the effects of increasing Gs protein on beta2-adrenergic receptor, Gs, and adenylyl cyclase interactions.

We have examined the effect of increased Gs protein levels on the abilities of three different beta2-agonists to induce GTP shifts and stimulate adenylyl cyclase response in an effort to investigate the kinetic association between the beta2-adrenergic receptor Gs and adenylyl cyclase. Agonist competition binding analysis and adenylyl cyclase concentration-response assays revealed that increases in Gs protein resulted in proportional increases in the areas of the GTP shift and adenylyl cyclase activity. Changes in the magnitude of the GTP shift were evaluated with a novel and straightforward approach for analyzing the GTP shift data that allowed us to determine the proportion of high agonist affinity binding receptor population and the apparent dissociation constant between the agonist bound receptor and Gs, regardless of the Gs protein level or the type of beta2-agonist. Using this method, we concluded that increased Gs results in the accumulation of the receptor population displaying high affinity towards agonist (HRGs) by increasing the number of receptor-Gs complexes (to a receptor:Gs protein ratio of about 0.7 at maximal Gs expression) without affecting the affinity between hormone bound receptor and Gs. Using the Gs protein levels determined with our novel analysis, we ran simulations using the theoretical shuttle model equation that relates the EC50 to available Gs. Fitting the simulations to experimental data required a receptor to catalytic unit ratio of 0.45 and revealed at least two distinct stages for beta2-agonist-stimulated adenylyl cyclase activity, namely, the activation of Gs by the beta2-adrenergic receptor (a step whose rate is dependent on the type of agonist used to stimulate activity), and the activation of adenylyl cyclase by active Gs (a step whose rate is independent of the type of agonist).

The stability of the agonist beta2-adrenergic receptor-Gs complex: evidence for agonist-specific states.

A restricted version of the ternary complex model for receptor-G protein complex formation has recently been proposed. Known as the two-state model, this model proposes that in the context of agonist and G protein interactions, only two thermodynamic states exist for the receptor: active (R*) and inactive (R). One form of this model suggests that only the R* state of the receptor is capable of interacting with and subsequently activating G proteins. We directly tested the kinetic aspects of a strict two-state receptor model in a cell line containing the native beta2-adrenergic receptor that is capable of inducing Gs expression. We examined adenylyl cyclase activity in the presence of limiting GTP levels and concluded that there exists a different rate of heterotrimer dissociation (i.e., HR*G yields HR* + G*) for different beta2-agonists. This finding is inconsistent with a strict two-state model in which R* is a characteristic of the receptor that is independent of the identity of the agonist. It implies that agonist activation of adenylyl cyclase is more complicated than a simple two-state model.