Maurice K.C. Ho

G z signaling: Emerging divergence from G i signaling

A large variety of neurotransmitters, hormones, and chemokines regulate cellular functions via cell surface receptors that are coupled to guanine nucleotide-binding regulatory proteins (G proteins) belonging to the Gi subfamily. All members of the Gi subfamily, with the sole exception of Gz, are substrates for the pertussis toxin ADP-ribosyl transferase. Gz also exhibits unique biochemical and regulatory properties. Initial portrayals of the cellular functions of Gz bear high resemblance to those of other Gi proteins both in terms of the receptors and effectors linked to Gz. However, recent discoveries have begun to insinuate a distinct role for Gz in cellular communication. Functional interactions of the α subunit of Gz (Gαz) with the NKR-P1 receptor, Gαz-specific regulator of G protein signaling, p21-activated kinase, G protein-regulated inducers of neurite outgrowth, and the Eya2 transcription cofactor have been demonstrated. These findings provide possible links for Gz to participate in cellular development, survival, proliferation, differentiation and even apoptosis. In this review, we have drawn a sketch of a signaling network with Gz as the centerpiece. The emerging picture is one that distinguishes Gz from other members of the Gi subfamily.

Gα 16/z Chimeras Efficiently Link a Wide Range of G Protein— Coupled Receptors to Calcium Mobilization

G protein—coupled receptors (GPCRs) represent a class of important therapeutic targets for drug discovery. The integration of GPCRs into contemporary high-throughput functional assays is critically dependent on the presence of appropriate G proteins. Given that different GPCRs can discriminate against distinct G proteins, a universal G protein adapter is extremely desirable. In this report, the authors evaluated two highly promiscuous Gα16/z chimeras, 16z25 and 16z44, for their ability to translate GPCR activation into Ca2+ mobilization using the fluorescence imaging plate reader (FLIPR) and aequorin. A panel of 24 Gs- or Gi-coupled receptors was examined for their functional association with the Gα16/z chimeras. Although most of the GPCRs tested were incapable of inducing Ca2+ mobilization upon their activation by specific agonists, the introduction of 16z25 or 16z44 allowed all of these GPCRs to mediate agonist-induced Ca 2+ mobilization. In contrast, only 16 of the GPCRs tested were capable of using Gα16 to mobilize intracellular Ca2+. Analysis of dose-response curves obtained with the δ-opioid, dopamine D1 , and Xenopus melatonin Mel1c receptors revealed that the Gα 16/z chimeras possess better sensitivity than Gα16 in both the FLIPR and aequorin assays. Collectively, these studies help to validate the promiscuity of the Gα16/z chimeras as well as their application in contemporary drug-screening assays that are based on ligand-induced Ca 2+ mobilization. (Journal of Biomolecular Screening 2003:39-49)

Gα14 links a variety of Gi‐ and Gs‐coupled receptors to the stimulation of phospholipase C

The bovine Gα14 is a member of the Gq subfamily of G proteins that can regulate phospholipase Cβ isoforms but the extent to which Gα14 recognizes different receptor classes is not known. Gα14 was cotransfected with a variety of receptors in COS‐7 cells, and agonist‐induced stimulation of phospholipase C was then measured. Activation of the type 2 but not type 1 somatostatin receptor in cells coexpressing Gα14 stimulated the accumulation of inositol phosphates; functional expression of both subtypes of somatostatin receptors was determined by the ability of somatostatin to inhibit cyclic AMP accumulation. Among the three opioid receptors (μ, δ, and κ), only the δ receptor was capable of stimulating IP formation when coexpressed with Gα14 in COS‐7 cells. A panel of Gi‐ and Gs‐linked receptors was screened for their ability to stimulate IP accumulation via Gα14. The adenosine A1, complement C5a, dopamine D1, D2 and D5, formyl peptide, luteinizing hormone, secretin, and the three subtypes of melatonin (mt1, MT2, and Xenopus) receptors were all incapable of activating Gα14, while the α2‐ and β2‐adrenoceptors were able to do so. Gα14‐mediated stimulation of phospholipase Cβ was agonist dose‐dependent. These data demonstrate that although Gα14 can interact with different classes of receptors, it is much less promiscuous than Gα15 or Gα16.

Regulation of transcription factors by heterotrimeric G proteins.

Lessons from viral hijacks of cells and cancer biology suggest that the activation of G protein-coupled receptors (GPCRs) often results in the modulation of various transcription factors and cofactors. Since drugs acting on GPCRs represent a significant portion of therapeutic agents currently in use, it is important to understand the actions of GPCRs on gene expression. GPCRs and their associated heterotrimeric G proteins are known to regulate gene transcription through complex signaling networks. The G protein-mediated signaling cascades have been extensively studied and accumulating evidence indicates that the four subfamilies of G proteins may utilize both common and unique pathways for transcriptional regulation. This review aims to provide a contemporary account of our understanding on the regulation of transcription factors by GPCRs, with a special emphasis on specific regulations of transcription factors such as STAT3 and NF-kappaB by individual G protein subfamilies. Functional impacts of the signal integration between different pathways and the contributions by other GPCR-interacting molecules will also be briefly discussed.

Astragaloside IV and Cycloastragenol Stimulate the Phosphorylation of Extracellular Signal-Regulated Protein Kinase in Multiple Cell Types

Two Chinese herb-derived small molecule telomerase activators, astragaloside IV (AG-IV) and cycloastragenol (CAG), have recently been shown to improve the proliferative response of CD8+ T lymphocytes from HIV-infected patients by upregulating telomerase activity. Here, we examined the signaling mechanism of AG-IV and CAG. Telomerase activity in human embryonic kidney HEK293 fibroblasts was increased upon treatment with increasing concentrations of AG-IV or CAG. Both compounds induced the phosphorylation of extracellular signal-regulated protein kinase (ERK) in a time- and dose-dependent manner in HEK293 cells and HEK-neo keratinocytes. AG-IV and CAG also stimulated ERK phosphorylation in other cell lines of lung, brain, mammary, endothelial, and hematopoietic origins. Use of selective inhibitors and dominant negative mutants revealed the involvement of c-Src, MEK (ERK kinase), and epidermal growth factor receptor in CAG-induced ERK phosphorylation. Our data indicate that AG-IV and CAG may exert their cellular effects through the activation of the Src/MEK/ERK pathway.

Synthesis of substituted N-[3-(3-methoxyphenyl)propyl] amides as highly potent MT2-selective melatonin ligands

A series of substituted N-[3-(3-methoxyphenyl)propyl] amides were synthesized and their binding affinities towards human melatonin MT(1) and MT(2) receptors were evaluated. It was discovered that a benzyloxyl substituent incorporated at C6 position of the 3-methoxyphenyl ring dramatically enhanced the MT(2) binding affinity and at the same time decreased MT(1) binding affinity.

Replacement of the α5 helix of Gα16 with Gαs-specific sequences enhances promiscuity of Gα16 toward Gs-coupled receptors

Abstract G16 can couple indiscriminately to a large number of G protein-coupled receptors (GPCRs), making it a prime candidate as a universal adaptor for GPCRs. In order to increase the promiscuity of Gα16, three chimeras incorporating increasing lengths of Gs-specific residues (25, 44 or 81 residues) into the C-terminus of Gα16 were constructed and named 16s25, 16s44 and 16s81, respectively. The chimeras were examined for their ability to mediate receptor-induced stimulation of phospholipase C (PLC) and Ca2+ mobilization. 16s25 was more effective than 16s44 and 16s81 at coupling to Gs-linked receptors. 16s25 coupled productively to 10 different Gs-coupled receptors examined and, for 50% of these receptors, 16s25-mediated PLC activities were higher than those mediated via Gα16. Similar results were observed for agonist-induced Ca2+ mobilizations. These results show that incorporating the α5 helix of Gαs into Gα16 can increase the promiscuity of 16s25 towards Gs-coupled receptors.

Gα16 activates Ras by forming a complex with tetratricopeptide repeat 1 (TPR1) and Son of Sevenless (SOS)

Many G protein-coupled receptors (GPCRs) are known to modulate cell growth and differentiation by stimulating the extracellular signal-regulated protein kinases (ERKs). In growth factor signaling, ERKs are typically stimulated through an elaborate network of modules consisting of adaptors, protein kinases, and the small GTPase Ras. The mechanism by which G protein signals tap into the ERK signaling pathway has thus far remain elusive. Members of the Gq family of G proteins, in particular Galpha16, have been shown to associate with tetratricopeptide repeat 1 (TPR1), an adaptor protein which preferentially binds to Ras. Here, we examined if TPR1 is indeed the missing link between Galpha16 signaling and Ras activation. Expression of Galpha16QL, a constitutively active mutant of Galpha16, in HEK 293 cells led to the formation of GTP-bound Ras and the subsequent phosphorylation of ERK. Likewise, stimulation of endogenou G16-coupled CCR1 chemokine receptors produced the same responses in human erythroleukemia cells. siRNA-mediated knockdown of TPR1 or expression of a dominant negative mutant of TPR1 effectively abolished the ability of Galpha16QL to induce Ras activation in HEK 293 cells. In contrast, these manipulations had no inhibitory effect on Galpha16QL induced activation of phospholipase Cbeta. Galpha16QL-induced phosphorylations of downstream targets including ERK, signal transducer and activator of transcription 3, and IkappaB kinase were significantly suppressed upon expression of the dominant negative mutant of TPR1. Furthermore, SOS2, a Ras guanine nucleotide exchange factor, was found to form a complex with TPR1 and Galpha16QL. Expression of SOS2 enhanced Galpha16QL-induced Ras activation and its subsequent signaling. Collectively, our results suggest that Galpha16 regulates multiple signaling pathways by activating Ras through its association with TPR1, but TPR1 is not required for Galpha16 to stimulate phospholipase Cbeta.

A Hematopoietic Perspective on the Promiscuity and Specificity of Gα16 Signaling

Gα16, a member of Gq subfamily, is expressed exclusively in hematopoietic cells, and its expression is highly modulated during lineage differentiation. Although functional redundancy within Gq subclass members has been observed in many established models, Gα16 possesses unique structural and biochemical properties not shared by other family members. Its broad receptor-coupling capacity and unique downstream binding partners and effectors allow the occurrence of both inositol lipid-dependent and -independent signals. Apart from its recognized biological functions in hematopoietic cell responses, the enlistment of complicated signaling pathways further signifies the importance of Gα16 in signal integration. This review aims to provide an updated appreciation and rational discussion of Gα16 signaling with regard to its promiscuity and specificity.

Identification of a stretch of six divergent amino acids on the alpha5 helix of Galpha16 as a major determinant of the promiscuity and efficiency of receptor coupling.

A broad repertory of G-protein-coupled receptors shows effective coupling with the haematopoietic G16 protein. In the present study, individual residues along the C-terminal alpha5 helix of Galpha16 were examined for their contributions in defining receptor-coupling specificity. Residues that are relatively conserved within, but diverse between, the subfamilies of cloned Galpha subunits were mutated into the corresponding Galpha(z) residues. Six G(i)-linked receptors with different coupling efficiencies to Galpha16 were examined for their ability to utilize the various Galpha16 mutants to mediate agonist-induced inositol phosphate accumulation and Ca2+ mobilization. Co-operative enhancements of receptor coupling were observed with chimaeras harbouring multiple mutations at Glu350, Lys357 and Leu364 of Galpha16. Mutation of Leu364 into isoleucine appeared to be more efficient in enhancing receptor recognition compared with mutations at the other two sites. Mutation of a stretch of six consecutive residues (362-367) lying towards the end of the alpha5 helix was found to broaden significantly the receptor-coupling profile of Galpha16, and the effect was mediated partly through interactions with the beta2-beta3 loop. These results suggested that a stretch of six distinctive residues at the alpha5 helix of Galpha16 is particularly important, whereas other discrete residues spreading along the alpha5 helix function co-operatively for determining the specificity of receptor recognition.