ABSTRACT
BACKGROUND AND PURPOSE 3-Hydroxy-octanoate, recently identified as a ligand for, the orphan GPCR, HCA(3), is of particular interest given its ability to treat lipid disorders and atherosclerosis. Here we demonstrate the pathway of HCA(3)-mediated activation of ERK1/2. EXPERIMENTAL APPROACH Using CHO-K1 cells stably expressing HCA(3) receptors and A431 cells, a human epidermoid cell line with high levels of endogenous expression of functional HCA(3) receptors, HCA(3)-mediated activation of ERK1/2 was measured by Western blot. KEY RESULTS HCA(3)-mediated activation of ERK1/2 was rapid, peaking at 5 min, and was Pertussis toxin sensitive. Our data, obtained by time course analyses in combination with different kinase inhibitors, demonstrated that on agonist stimulation, HCA(3) receptors evoked ERK1/2 activation via two distinct pathways, the PLC/PKC pathway at early time points (≤ 2 min) and the MMP/ epidermal growth factor receptor (EGFR) transactivation pathway with a maximum response at 5 min. Furthermore, our present results also indicated that the ßγ-subunits of the G(i) protein play a critical role in HCA(3)-activated ERK1/2 phosphorylation, whereas ß-arrestins and Src were not required for ERK1/2 activation. CONCLUSIONS AND IMPLICATIONS We have described the molecular mechanisms underlying the coupling of human HCA(3) receptors to the ERK1/2 MAP kinase pathway in CHO-K1 and A431 cells, which implicate the G(i) protein-initiated, PLC/PKC -and platelet-derived growth factor receptor/EGFR transactivation-dependent pathways. These observations may provide new insights into the pharmacological effects and the physiological functions modulated by the HCA(3)-mediated activation of ERK1/2.
Subject(s)
ErbB Receptors/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Protein Kinase C/metabolism , Receptors, G-Protein-Coupled/metabolism , Receptors, Nicotinic/metabolism , Animals , Arrestins/metabolism , CHO Cells , Calcium/metabolism , Cricetinae , Cricetulus , Cyclic AMP/metabolism , Dipeptides/pharmacology , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , HEK293 Cells , Humans , MAP Kinase Kinase 1/metabolism , MAP Kinase Kinase 2/metabolism , MAP Kinase Signaling System/drug effects , Metalloendopeptidases/antagonists & inhibitors , Metalloendopeptidases/metabolism , beta-ArrestinsABSTRACT
BACKGROUND AND PURPOSE: The cannabinoid CB(1) receptor is primarily thought to be functionally coupled to the G(i) form of G proteins, through which it negatively regulates cAMP accumulation. Here, we investigated the dual coupling properties of CB(1) receptors and characterized the structural determinants that mediate selective coupling to G(s) and G(i). EXPERIMENTAL APPROACH: A cAMP-response element reporter gene system was employed to quantitatively analyze cAMP change. CB(1)/CB(2) receptor chimeras and site-directed mutagenesis combined with functional assays and computer modelling were used to determine the structural determinants mediating selective coupling to G(s) and G(i). KEY RESULTS: CB(1) receptors could couple to both G(s)-mediated cAMP accumulation and G(i)-induced activation of ERK1/2 and Ca(2+) mobilization, whereas CB(2) receptors selectively coupled to G(i) and inhibited cAMP production. Using CB(1)/CB(2) chimeric receptors, the second intracellular loop (ICL2) of the CB(1) receptor was identified as primarily responsible for mediating G(s) and G(i) coupling specificity. Furthermore, mutation of Leu-222 in ICL2 to either Ala or Pro switched G protein coupling from G(s) to G(i), while to Ile or Val led to balanced coupling of the mutant receptor with G(s) and G(i) . CONCLUSIONS AND IMPLICATIONS: The ICL2 of CB(1) receptors and in particular Leu-222, which resides within a highly conserved DRY(X)(5) PL motif, played a critical role in G(s) and G(i) protein coupling and specificity. Our studies provide new insight into the mechanisms governing the coupling of CB(1) receptors to G proteins and cannabinoid-induced tolerance.
Subject(s)
GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , GTP-Binding Protein alpha Subunits, Gs/metabolism , Protein Binding/genetics , Protein Interaction Domains and Motifs , Receptor, Cannabinoid, CB1/metabolism , 3T3 Cells , Adenylyl Cyclases/drug effects , Animals , CHO Cells , COS Cells , Calcium/metabolism , Cell Line, Transformed , Chlorocebus aethiops , Cricetinae , Cricetulus , Cyclic AMP/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , HEK293 Cells , Humans , Mice , Mutagenesis, Site-Directed/methods , Receptor, Cannabinoid, CB1/genetics , Receptor, Cannabinoid, CB2/genetics , Receptor, Cannabinoid, CB2/metabolismABSTRACT
The authors describe the design and validation of a competitive RT-PCR method for the efficient and reproducible quantitation of mRNA molecules of IFN-gamma, TNF-alpha, IL-4 and IL-10 in mouse spleen RNA extracts. Before being subjected to RT-PCR, the RNA extracts were supplemented with internal control RNAs (IC-RNAs), which were constructed by inserting DNA fragments in the cDNA of the respective cytokines. The efficiency of amplification of the target and the IC-RNA was shown to remain equal over a wide range of cycle numbers. Reproducibility was such that differences in mRNA contents that were greater than 17% could be detected between two RNA samples run in parallel. Normal mouse spleen tissue was found to contain 10(7)-10(8) molecules of TNF-alpha, IFN-gamma, IL-4 and IL-10 mRNA per micrograms total RNA extracted. Injection of animals with anti-CD3 antibody, a well-known cytokine inducer, resulted in a moderate increase in TNF-alpha and IL-10 mRNA levels (14- and 24-fold, respectively), and in a substantially greater increase in the levels of mRNA for IL-4 and IFN-gamma (199- and 851-fold, respectively). These results demonstrate an accurate and reliable quantitation of cytokine mRNA levels in animal tissues.
