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Altered regulation of dopamine D(2) receptors is implicated in addiction, schizophrenia and movement disorders, as well as lactotroph growth and regulation. Dopamine D(2S) and dopamine D(2L) receptors are alternately-spliced variants that differ by 29 amino acids in the third intracellular (i3) domain and display different sensitivity to desensitization by protein kinase C (PKC). In the present studies we determined the specific phosphorylation sites on the dopamine D(2S) receptor that confer PKC-mediated desensitization. In dopamine D(2L) receptors, we identified a PKC pseudosubstrate site responsible for the relative insensitivity of the receptor to PKC-induced uncoupling. In transiently transfected Ltk(-) fibroblast cells, 2-min preactivation of PKC with 12-O-tetradecanoyl 4beta-phorbol 13alpha-acetate (TPA) completely inhibited calcium mobilization induced by the dopamine D(2S) receptor, but not the dopamine D(2L) variant. Point mutation of i3 PKC sites Ser228/229Gly rendered the dopamine D(2S) receptor resistant to PKC action, with lesser effects of other Ser and Thr mutations. Inactivation of the PKC pseudosubstrate motif in the dopamine D(2L) receptor sensitized the receptor to PKC, and this was reversed by mutation of i3 PKC sites Ser228/229. A phospho-specific antibody generated against phospho-Ser228/229 demonstrated PKC-induced phosphorylation at these sites of dopamine D(2S), but not D(2L) receptors, in Ltk(-) cells. Conversely, the pseudosubstrate dopamine D(2L) receptor mutant displayed PKC-induced phosphorylation at Ser228/229, which was abolished when these sites were mutated. Similar phosphorylation results were observed using GH4 cells stably transfected with dopamine D(2) receptors and mutants. Thus the relative location of phosphorylation and pseudosubstrate sites provides an important determinant substrate sensitivity to PKC.

Although they have distinct functions, the signaling of dopamine-D(2) receptor short and long isoforms (D(2)S and D(2)L) is virtually identical. We compared inhibitory regulation of extracellular signal-regulated kinases (ERK1/2) in GH4 pituitary cells separately transfected with these isoforms. Activation of rat or human dopamine-D(2)S, muscarinic or somatostatin receptors inhibited thyrotropin-releasing hormone-induced ERK1/2 phosphorylation, while the D(2)L receptor failed to inhibit this response. In order to address the structural basis for the differential signaling of D(2)S and D(2)L receptors, we examined the D(2)L-SS mutant, in which a protein kinase C (PKC) pseudosubstrate site that is present in the D(2)L but not D(2)S receptor was converted to a consensus PKC site. In transfected GH4 cells, the D(2)L-SS mutant inhibited thyrotropin-releasing hormone-induced ERK1/2 phosphorylation almost as strongly as the D(2)S receptor. A D(2)S-triple mutant that eliminates PKC sites involved in D(2)S receptor desensitization also inhibited ERK1/2 activation. Similarly, in striatal cultures, the D(2)-selective agonist quinpirole inhibited potassium-stimulated ERK1/2 phosphorylation, indicating the presence of this pathway in neurons. In conclusion, the D(2)S and D(2)L receptors differ in inhibitory signaling to ERK1/2 due to specific residues in the D(2)L receptor alternatively spliced domain, which may account for differences in their function in vivo.

We have shown previously that responses to lysophosphatidic acid (LPA) in Xenopus oocytes exhibit pronounced rapid homologous desensitization mediated by Go family of G-proteins (Itzhaki-Van Ham et al., 2004, J Cell Physiol, 200: 125-133). The present study was aimed at examining the involvement of Go G-proteins in rapid heterologous desensitization of native and expressed G-protein-coupled receptors in Xenopus oocytes. Threshold stimulation of the native lysophosphatidic acid receptors (LPA-Rs) induced about 50% rapid desensitization of responses evoked by stimulation of either native trypsin or expressed M1-muscarinic cholinergic receptors (M1-Rs). Similarly, threshold stimulation of expressed M1-Rs or thyrotropin-releasing hormone receptors induced 40% rapid desensitization of responses to LPA. Inactivation of all Gi/o G-proteins with pertussis toxin (PTX) completely abolished rapid heterologous desensitization in all protocols. Depletion of either Galphao or Galphao1 by antisense oligodeoxynucleotides targeted at either member of the Galphao family decreased or completely abolished rapid heterologous desensitization. Expression of two dominant negative mutants of the human Galphao family, highly homologous to oocyte Galphao species, either decreased or virtually abolished rapid desensitization. Homologous and heterologous desensitizations of the LPA response were non-additive and proceeded, apparently, via the same pathway. We conclude that Go G-proteins mediate both homologous and heterologous rapid desensitization of responses mediated by G-protein-coupled receptors (GPCRs) coupled to the phosphoinositide phospholipase C-inositol 1,4,5-trisphosphate-Ca(2+) (PI-PLC-InsP(3)-Ca(2+)) pathway in Xenopus oocytes., ((c) 2005 Wiley-Liss, Inc.)

Native Xenopus oocytes exhibit dose-dependent depolarizing current responses to lysophosphatidic acid (LPA), with EC50 = 0.18 microM. Responses to LPA were subject to pronounced rapid desensitization. When oocytes were challenged with 5 nM LPA, the response was <10% of the maximal. Subsequent addition of 0.5 microM LPA resulted in 50-70% desensitization, when compared to naïve controls. Injection of antisense oligodeoxyoligonucleotides (ASODNs) targeted at either of the two endogenous LPA receptors inhibited the LPA response by approximately 50%, but did not alter the degree of rapid desensitization. To study the involvement of G-proteins in rapid homologous desensitization of responses to LPA, we selectively depleted native G-proteins by injection of specific ASDONs. Injection of ASDONs targeted at Galphaq family mRNAs (mainly Galpha11) reduced the response to 0.5 microM LPA by 50%. ASDONs targeted at either Galphao or Galphao1 caused a large decrease in the amount of their cognate mRNAs and the Galphao family proteins, while the response to LPA was inhibited by up to 30%. Injection of ASDONs targeted at Galphao1 mRNA decreased rapid desensitization from 69 to 23%, while pertussis toxin (PTX) completely abolished it. Expression of two dominant negative mutants of the human Galphao family homologs either decreased or virtually abolished rapid desensitization. Microinjection of CaCl(2) demonstrated that 50% of rapid desensitization could be attributed to inhibition of Ca(2+) activation of chloride channels. We propose that the apparent degenerate coupling of different G-proteins to LPA receptors in Xenopus oocytes actually serves both the generation of the response (by Gq and Go G-protein families) and its desensitization (mostly by Go G-protein family)., (Copyright 2004 Wiley-Liss, Inc.)