Multiple signaling routes involved in the regulation of adenylyl cyclase and extracellular regulated kinase by dopamine D(2) and D(3) receptors.

Most G protein coupled receptors (GPCR) regulate multiple cellular processes by coupling to more than one kind of G protein. Furthermore, recent studies have reported G protein-independent/ß-arrestin-dependent signaling pathway for some GPCRs. Dopamine D(2) and D(3) receptors (D(2)R, D(3)R), the major targets of currently used antipsychotic drugs, are co-expressed in some of the same dopaminergic neurons and regulate the same overlapping effectors. However, the specific subunits of G proteins that regulate each signaling pathway are not clearly identified. In addition, the existence of ß-arrestin-dependent/G protein-independent signaling is not clear for these receptors. In this study, we determined the G protein subtypes and ß-arrestin dependency involved in the signaling of D(2)R and D(3)R, which was measured by inhibition of adenylyl cyclase and extracellular signal-regulated kinase (ERK) activation. For the inhibition of cAMP production in HEK-293 cells, D(2)R used the Gαo subunit but D(3)R used the ßγ subunit of Gi family proteins. For the regulation of ERK activation, D(2)R used the α subunits of Gi/o proteins both in HEK-293 cells and COS-7 cells, but D(3)R used Gαo and Gßγ in HEK-293 cells and COS-7 cells, respectively. ß-Arrestin-dependent/G protein-independent ERK activation was not observed for both D(2)R and D(3)R. Agonist-induced ß-arrestin translocation was observed with D(2)R but not with D(3)R, and ß-arrestins exerted inhibitory influences on G protein-dependent ERK activation by D(2)R, but not D(3)R. These results show that the D(2)R and D(3)R, which have overlapping cellular expressions and functional roles, employ distinct G protein subunits depending on the cell types and the effectors they control.

RGS4 exerts inhibitory activities on the signaling of dopamine D2 receptor and D3 receptor through the N-terminal region.

Dopamine D(2) receptor and D(3) receptor (D(2)R and D(3)R) are the major targets for current antipsychotic drugs, and their proper regulation has pathological and pharmacological significance. This study was conducted to understand the functional roles and molecular mechanisms of RGS proteins (RGS2, RGS4, and RGS9-2) on the signaling of D(2)R and D(3)R. RGS proteins were co-expressed with D(2)R and D(3)R in HEK-293 cells. The protein interactions between RGS proteins and D(2)R/D(3)R, and effects of RGS proteins on the internalization, signaling, and desensitization of D(2)R/D(3)R were determined. In addition, the RGS4 proteins were subdivided into N-terminal region, RGS domain, and the C-terminal region, and the specific subdomain of RGS4 protein involved in the regulation of the signaling of D(2)R/D(3)R was determined. All of RGS proteins we tested interacted with D(2)R/D(3)R. RGS4 exerted potent inhibitory activities on the signaling of D(2)R/D(3)R. RGS9-2 exerted selective but moderate inhibitory activity on D(3)R and the internalization of D(2)R. RGS2 had no effect. The N-terminal domain of RGS4 was involved in its interaction with D(2)R and D(3)R and was required for the inhibitory activity of the RGS domain. The study for the first time showed that RGS4 is the major RGS protein which interacts through the N-terminal region and exerts potent inhibitory activities on the signaling of D(2)R and D(3)R.

A thaumatin-like gene in nonclimacteric pepper fruits used as molecular marker in probing disease resistance, ripening, and sugar accumulation.

During pepper (Capsicum annuum) fruit ripening, the ripe fruit interaction with the anthracnose fungus, Colletotrichum gloeosporioides, is generally incompatible. However, the unripe fruit can interact compatibly with the fungus. A gene, designated PepTLP (for pepper thaumatin-like protein), was isolated and characterized by using mRNA differential display. The PepTLP gene encodes a protein homologous to other thaumatin-like proteins and contains 16 conserved cysteine residues and the consensus pattern of thaumatin. PepTLP gene expression is developmentally regulated during ripening. The accumulation of PepTLP mRNA and PepTLP protein in the incompatible interaction was higher than that in the compatible one. Furthermore, PepTLP gene expression was stimulated by both jasmonic acid treatment and wounding during ripening, but by wounding only in the unripe fruit. Immunolocalization studies showed that it is localized to the intercellular spaces among cortical cells. The expression of the PepTLP gene upon fungal infection was a rise from the early-breaker fruit. The development of anthracnose became significantly prevented with beginning of fruit ripening, and the sum total of sugar accumulation increased. The results suggest that the PepTLP gene can be used as a molecular marker in probing for disease resistance, ripening, and sugar accumulation in nonclimacteric pepper fruits.