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Rho is a major small GTP-binding protein that is involved in the regulation of various cell functions, including proliferation and cell migration, through activation of multiple signaling molecules in various types of cells. We studied its roles in synovial fibroblasts (SFs) in patients with rheumatoid arthritis (RA) and clarified its relevance to RA synovitis, with the following results. 1)We found that the thrombin receptor was overexpressed on RA synovial fibroblasts (RA SFs) and that thrombin induced a marked proliferation and progression of the cell cycle to the S phase in these cells. 2)We also found that thrombin efficiently activated Rho. 3)Rho activation and proliferation and the progression of the cell cycle to the S phase were completely blocked by p115RGS (an N-terminal regulator of the G-protein signaling domain of p115RhoGEF) and by the C-terminal fragments of Galpha13 (an inhibitor of the interaction of receptors with G13). 4)Thrombin induced the secretion of IL-6 by RA SFs, but this action was blocked by p115RGS or Galpha13. Our findings show that the actions of thrombin on the proliferation of RA SFs, cell-cycle progression to the S phase, and IL-6 secretion were mainly mediated by the G13 and RhoGEF pathways. These results suggest that p115RGS and Galpha13 could be potent inhibitors of such functions. A rational design of future therapeutic strategies for RA synovitis could perhaps include the exploitation of the Rho pathway to directly reduce the growth of synovial cells.

G protein-coupled receptors (GPCRs) are the most famous target proteins for medicinal drugs. So far, heterogeneity of GPCRs is mainly focused on genetic variation. However, it has been reported that the structure and function of GPCRs are modified by several mechanisms after translation. RNA editing introduces the amino acid different from that encoded in genome by changing the nucleotide. Dimer formation is another example of how heterogeneity is produced. Many receptors form homo- or hetero-dimers, and obtain different function from original receptors. Receptors are regulated by several means to modulate stimulation strength. Receptor subtype is often differentially regulated by receptor kinases and/or second messenger-regulated kinases. There is a new type of receptor that shows a novel structural feature, a long amino terminal region belonging to class B seven transmembrane receptors. The physiological function of this class of receptor is assumed to play a role in cell-cell communication. This novel structural feature may directly link GPCR to the cytoskeleton. These mechanisms to produce functional and structural heterogeneity may explain how cells evoke different responses in different tissues or cells upon the same stimulation. Thus, the post-translational mechanism to produce heterogeneity provides additional flexibility when cells respond to one extracellular stimulus.

It is generally thought that Galpha(12) and Galpha(13)-induced responses are exclusively mediated by small G protein Rho. However, Galpha(12) and Galpha(13) elicit divergent cellular responses: phospholipase C-epsilon activation, phospholipase D activation, cytoskeletal change, oncogenic response, apoptosis, MAP kinase activation and Na/H-exchange activation. In addition to Rho activation through RhoGEF, it has been recently demonstrated that Galpha(12) and Galpha(13) interact with several proteins and regulate their activities. However, physiological importance of the interaction of Galpha(12) and Galpha(13) with these proteins has not fully established. I summarize the recent progress of Galpha(12) and Galpha(13)-mediated signaling cascade.

At the beginning, beta-arrestin is believed to regulate desensitization of G protein-coupled receptors. However, it becomes apparent that beta-arrestin is involved in not only regulation but also signal transduction by identification of many proteins interacting with beta-arrestin. So far, the model of regulation and signaling of G protein-coupled receptor is built on beta 2-adrenergic receptor. However, this model does not apply to all G protein-coupled receptors. The differential regulation and signaling between beta 1- and beta 2-adrenergic receptors is presented.

In the present study, we examined the roles of G(12), G(13), G(q), and G(i) in endothelin-1-induced hypertrophic responses. Endothelin-1 stimulation activated extracellular signal-regulated kinase (ERK) and c-Jun NH(2)-terminal kinase (JNK) in cultured rat neonatal myocytes. The activation of JNK, but not ERK, was inhibited by the expression of carboxyl terminal regions of G alpha(12) and G alpha(13). JNK activation was also inhibited by expression of the G alpha(12)/G alpha(13)-specific inhibitor regulator of G protein signaling (RGS) domain of p115RhoGEF and the G alpha(q)-specific inhibitor RGS domain of the G protein-coupled receptor kinase 2 (GRK2-RGS). JNK activation was not, however, inhibited by expression of the carboxyl terminal region of G protein-coupled receptor kinase 2 (GRK2-ct), which is a G beta gamma-sequestering polypeptide. Additionally, JNK activation but not ERK activation was inhibited by the expression of C3 exoenzyme that inactivates small GTPase Rho. These results suggest that JNK activation by G alpha(12), G alpha(13), and G alpha(q) is involved in Rho. On the other hand, ERK activation was inhibited by pertussis toxin treatment, the receptor-G(i) uncoupler, and GRK2-ct. Thus, ERK was activated by G alpha(i)- and G beta gamma-dependent pathways. These results clearly demonstrate that differential pathways activate JNK and ERK.

Beta(3)-adrenoceptor is the predominant beta-adrenoceptor in adipocytes and has drawn much attention during the investigation for anti-obesity and antidiabetes therapeutics. Thirteen new compounds have been evaluated for their potencies and efficacies as beta(3)-adrenoceptor agonists on human beta(3)-adrenoceptor expressed in COS-7 and Chinese hamster ovary (CHO) cells using radioligand binding assay and cyclic AMP (cAMP) accumulation assay. Phenoxypropanolamine derivatives, SWR-0334NA (([E)-[4-[5-[(3-phenoxy-2-hydroxypropyl)amino]-2-pentene-3-yl] phenoxy]acetic acid sodium salt), SWR-0335SA ((E)-[4-[5-[(3-phenoxy-2-hydroxypropyl)amino]-2-pentene-3-yl] phenoxy] acetic acid ethanedioic acid), SWR-0342SA (S-(Z)-[4-[[1-[2-[(2-hydroxy-3-phenoxypropyl)]amino] ethyl]-1-propenyl]phenoxy] acetic acid ethanedioic acid), SWR-0348SA-SITA ((E)-[4-[5-[(3-phenoxy-2-hydroxypropyl)amino]-2-hexene-3-yl] phenoxy]acetic acid ethanedioic acid) and SWR-0361SA ((E)-N-methyl[4-[5-[(3-phenoxy-2-hydroxypropyl)amino]-2-pentene-3-yl]phenoxy]acetoamide ethanedioic acid) showed higher agonistic activity for the beta(3)-adrenoceptor. Among the compounds tested, SWR0334NA exhibited full agonist activity (%E(max) = 100.26) despite its lower binding affinity (pK(I) = 6.11). Compounds SWR-0338SA ((E)-[4-[5-[(2-phenyl-2-hydroxyethyl)amino]-2-pentene-3-yl] phenoxy]acetic acid ethanedioic acid), SWR-0339SA (S-(E)-[4-[5-[(3-phenoxy-2-hydroxypropyl)amino]-2-pentene-3-yl] phenoxy] acetic acid ethanedioic acid), SWR-0345HA ((E)-2-methyl-3-[4-[2-(2-phenyl-2-hydroxyethyl-amino)ethoxy] phenyl]-2-propenoic acid ethyl ester hydrochloride), SWR-0358SA ((E)-(2-methoxyethyl)-[4-[5-[(3-phenoxy-2-hydroxypropyl) amino]-2-pentene-3-yl]phenoxy]acetoamide ethanedioic acid) and SWR-0362SA ((E)-1-[[[4-[5-[(3-phenoxy-2-hydroxypropyl)amino]-2-pentene-3-yl]phenoxy]acetyl]carbonyl]piperidine ethanedioic acid) had moderate agonistic activity and were phenethylamine and phenoxypropanolamine derivatives. Compounds SWR-0065HA ([4-[2-[3-[[(3,4-dihydro-4-oxo-[1,2,4]-triazino(4,5-a)indol)-lyl]oxy]-2-hydroxypropylamino]ethoxy]phenyl]acetic acid methyl ester hydrochloride), SWR-0098NA ((E)-[4-[3-[(2-phenyl-2-hydroxyethyl)amino]-1-butenyl] phenoxy]acetic acid sodium salt) and SWR-0302HA ([4-[[4-[2-(3-chlorophenoxy-2-hydroxypropyl)amino]-E-2-butenyl]oxy]phenoxy]acetic acid hydrochloride) had very low binding affinity towards beta(3)-adrenoceptors and they did not induce cAMP accumulation. We concluded that compounds SWR-0334NA, SWR-0335SA, SWR-0342SA, SWR-0348SA-SITA and SWR-0361SA were potential agonists of human beta(3)-adrenoceptor. Further investigation on their selectivity towards beta(3)-adrenoceptor could be useful for the exploration of the physiological properties of the beta(3)-adrenoceptor.

In neonatal cardiomyocytes, activation of the G(q)-coupled alpha(1)-adrenergic receptor (alpha(1)AR) induces hypertrophy by activating mitogen-activated protein kinases, including c-Jun NH(2)-terminal kinase (JNK). Here, we show that JNK activation is essential for alpha(1)AR-induced hypertrophy, in that alpha(1)AR-induced hypertrophic responses, such as reorganization of the actin cytoskeleton and increased protein synthesis, could be blocked by expressing the JNK-binding domain of JNK-interacting protein-1, a specific inhibitor of JNK. We also identified the classes and subunits of G proteins that mediate alpha(1)AR-induced JNK activation and hypertrophic responses by generating several recombinant adenoviruses that express polypeptides capable of inhibiting the function of specific G-protein subunits. alpha(1)AR-induced JNK activation was inhibited by the expression of carboxyl terminal regions of Galpha(q), Galpha(12), and Galpha(13). JNK activation was also inhibited by the Galpha(q/11)- or Galpha(12/13)-specific regulator of G-protein signaling (RGS) domains and by C3 toxin but was not affected by treatment with pertussis toxin or by expression of the carboxyl terminal region of G protein-coupled receptor kinase 2, a polypeptide that sequesters Gbetagamma. alpha(1)AR-induced hypertrophic responses were inhibited by Galpha(q/11)- and Galpha(12/13)-specific RGS domains, C3 toxin, and the carboxyl terminal region of G protein-coupled receptor kinase 2 but not by pertussis toxin. Activation of Rho was inhibited by carboxyl terminal regions of Galpha(12) and Galpha(13) but not by Galpha(q). Our findings suggest that alpha(1)AR-induced hypertrophic responses are mediated in part by a Galpha(12/13)-Rho-JNK pathway, in part by a G(q/11)-JNK pathway that is Rho independent, and in part by a Gbetagamma pathway that is JNK independent.

(-)-1-(3,4-Dimethoxyphenetylamino)-3-(3,4-dihydroxy)-2-propanol [(-)-RO363] is a highly selective beta(1)-adrenergic receptor (beta(1)AR) agonist. To study the binding site of beta(1)-selective agonist, chimeric beta(1)/beta(2)ARs and Ala-substituted beta(1)ARs were constructed. Several key residues of beta(1)AR [Leu(110) and Thr(117) in transmembrane domain (TMD) 2], and Phe(359) in TMD 7] were found to be responsible for beta(1)-selective binding of (-)-RO363, as determined by competitive binding. Based on these results, we built a three-dimensional model of the binding domain for (-)-RO363. The model indicated that TMD 2 and TMD 7 of beta(1)AR form a binding pocket; the methoxyphenyl group of N-substituent of (-)-RO363 seems to locate within the cavity surrounded by Leu(110), Thr(117), and Phe(359). The amino acids Leu(110) and Phe(359) interact with the phenyl ring of (-)-RO363, whereas Thr(117) forms hydrogen bond with the methoxy group of (-)-RO363. To examine the interaction of these residues with beta(1)AR in an active state, each of the amino acids was changed to Ala in a constitutively active (CA)-beta(1)AR mutant. The degree of decrease in the affinity of CA-beta(1)AR for (-)-RO363 was essentially the same as that of wild-type beta(1)AR when mutated at Leu(110) and Thr(117). However, the affinity was decreased in Ala-substituted mutant of Phe(359) compared with that of wild-type beta(1)AR. These results indicated that Leu(110) and Thr(117) are necessary for the initial binding of (-)-RO363 with beta(1)-selectivity, and interaction of Phe(359) with the N-substituent of (-)-RO363 in an active state is stronger than in the resting state.

In rat neonatal myocytes, a constitutively active G alpha(q) causes cellular injury and apoptosis. However, stimulation of the alpha(1)-adrenergic receptor, one of the G(q) protein-coupled receptors, with phenylephrine for 48 h causes little cellular injury and apoptosis. Expression of the G beta gamma-sequestering peptide beta ARK-ct increases the phenylephrine-induced cardiac injury, indicating that G beta gamma released from G(q) counteracts the G alpha(q)-mediated cellular injury. Stimulation with phenylephrine activates extracellular signal-regulated kinase (ERK) and Akt, and activation is significantly blunted by beta ARK-ct. Inhibition of Akt by inhibitors of phosphatidylinositol 3-kinase increases the cellular injury induced by phenylephrine stimulation. In contrast to the inhibition of Akt, inhibition of ERK does not affect the phenylephrine-induced cardiac injury. These results suggest that G beta gamma released from G(q) upon alpha(1)-adrenergic receptor stimulation activates ERK and Akt. However, activation of Akt but not ERK plays an important role in the protection against the G alpha(q)-induced cellular injury and apoptosis.

We have examined the functional significance of the naturally occurring mutation at position 64 of human beta3-adrenergic receptor (beta3AR), which changes the amino acid from tryptophan to arginine (W64R-beta3AR). The affinities of betaAR agonists for W64R-beta3AR expressed in COS-7 cells were not significantly different from those for wild type beta3AR. When two receptors are expressed at various expression levels, and stimulated with CGP12177A, they showed essentially the same EC50 values and maximal responses. Overexpression of Gi and Go, or the treatment with pertussis toxin did not affect the agonist-induced cAMP response, suggesting that Gi and Go did not contribute to the beta3AR-induced cAMP response. However, the enhanced cAMP response was observed when W64R-beta3AR was coexpressed with the adenylyl cyclase type III isoform, and stimulated by CGP12177A and isoproterenol. These results indicate that the cAMP response of W64R-beta3AR can be enhanced under the particular condition that adenylyl cyclase type III was coexpressed.

Extracellular information is translated into the cellular responses by various signal transduction systems such as G protein-coupled receptor-linked and protein tyrosine kinase receptor-mediated pathways. The changes or modifications of theses signaling proteins cause uncontrolled transformation or cell death. One of the features of the signaling molecules is that these proteins contain the domains or motifs for protein-protein or protein-lipid interactions governed by several amino acids. Therefore, these amino acids may become promising targets for the drug design. In contrast with intracellular molecules, the receptors located on cellular surface are still the best candidate for the drugs. The future analysis of cellular signal transduction system allows us to develop new drugs that cannot cause serious side effects in future.