Yurii V. Mukhin

Multiplicity of mechanisms of serotonin receptor signal transduction

The serotonin (5-hydroxytryptamine, 5-HT) receptors have been divided into 7 subfamilies by convention, 6 of which include 13 different genes for G-protein-coupled receptors. Those subfamilies have been characterized by overlapping pharmacological properties, amino acid sequences, gene organization, and second messenger coupling pathways. Post-genomic modifications, such as alternative mRNA splicing or mRNA editing, creates at least 20 more G-protein-coupled 5-HT receptors, such that there are at least 30 distinct 5-HT receptors that signal through G-proteins. This review will focus on what is known about the signaling linkages of the G-protein-linked 5-HT receptors, and will highlight some fascinating new insights into 5-HT receptor signaling.

The recombinant 5-HT1A receptor: G protein coupling and signalling pathways.

The 5‐hydroxytryptamine 5‐HT1A receptor was one of the first G protein coupled receptors whose cDNA and gene were isolated by molecular cloning methods. Transfection of the cDNA of this receptor into cells previously bearing no 5‐HT receptors has resulted in the acquisition of large amounts of information regarding potential signal transduction pathways linked to the receptor, correlations of receptor structure to its various functions, and pharmacological properties of the receptor. Transfection studies with the 5‐HT1A receptor have generated critical new information that might otherwise have been elusive. This information notably includes the discovery of unsuspected novel signalling linkages, the elucidation of the mechanisms of receptor desensitization, the refinement of models of the receptor pharmacophore, and the development of silent receptor antagonists, among others. The current review summarizes the most important studies of the recombinant 5‐HT1A receptor in the decade since the identificiation of its cDNA.

Serotonin 5-HT1A receptor-mediated Erk activation requires calcium/calmodulin-dependent receptor endocytosis.

Many receptors that couple to heterotrimeric guanine nucleotide-binding (G) proteins mediate rapid activation of the mitogen-activated protein kinases, Erk1 and Erk2. The Gi-coupled serotonin (5-hydroxytryptamine (5-HT)) 5-HT1A receptor, heterologously expressed in Chinese hamster ovary or human embryonic kidney 293 cells, mediated rapid activation of Erk1/2 via a mechanism dependent upon both Ras activation and clathrin-mediated endocytosis. This activation was attenuated by chelation of intracellular Ca2+ and Ca2+/calmodulin (CAM) inhibitors or the CAM sequestrant protein calspermin. The CAM-dependent step in the Erk1/2 activation cascade is downstream of Ras activation, because inhibitors of CAM antagonize Erk1/2 activation induced by constitutively activated mutants of Ras and c-Src but not by constitutively activated mutants of Raf and MEK (mitogen and extracellular signal-regulated kinase). Inhibitors of the classical CAM effectors myosin light chain kinase, CAM-dependent protein kinases II and IV, PP2B, and CAM-sensitive phosphodiesterase had no effect upon 5-HT1A receptor-mediated Erk1/2 activation. Because clathrin-mediated endocytosis was required for 5-HT1A receptor-mediated Erk1/2 activation, we postulated a role for CAM in receptor endocytosis. Inhibition of receptor endocytosis by use of sequestration-defective mutants of β-arrestin1 and dynamin attenuated 5-HT1Areceptor-stimulated Erk1/2 activation. Inhibition of CAM prevented agonist-dependent endocytosis of epitope-tagged 5-HT1A receptors. We conclude that CAM-dependent activation of Erk1/2 through the 5-HT1A receptor reflects its role in endocytosis of the receptor, which is a required step in the activation of MEK and subsequently Erk1/2.

Role for 18:1 Lysophosphatidic Acid as an Autocrine Mediator in Prostate Cancer Cells

Lysophosphatidic acid (LPA) is a lipid mediator that may play an important role in growth and survival of carcinomas. In this study, LPA production and response were characterized in two human prostate cancer (CaP) cell lines: PC-3 and Du145. Bombesin, a neuroendocrine peptide that is mitogenic for CaP cells, stimulated focal adhesion kinase phosphorylation and activated the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway. Similar responses were elicited by 18:1 LPA (oleoyl-LPA). Studies using radioisotopic labeling revealed that both PC-3 and Du145 generate LPA and that LPA production is increased by bombesin. The kinetics of bombesin-induced phospholipase D activation and LPA production were similar. Using electrospray ionization mass spectrometry, 18:1 LPA was found to be an abundant LPA species in CaP cell medium. Structure activity studies of acyl-LPAs revealed that 18:1 LPA is most efficacious for activation of extracellular signal-regulated kinase and phospholipase D in CaP cells. Incubation with 18:1 LPA caused homologous desensitization of LPA response, whereas bombesin caused heterologous desensitization. LPA was present at nanomolar levels in medium from bombesin-treated cells. LPA extracted from the medium induced calcium mobilization in CaP cells. These results demonstrate that bioactive LPA is generated by CaP cells in response to a mitogen and suggest that 18:1 LPA can act as an autocrine mediator.

ERK Is Regulated by Sodium-Proton Exchanger in Rat Aortic Vascular Smooth Muscle Cells

The purposes of this study were to test 1) the relationship between two widely studied mitogenic effector pathways, and 2) the hypothesis that sodium-proton exchanger type 1 (NHE-1) is a regulator of extracellular signal-regulated protein kinase (ERK) activation in rat aortic smooth muscle (RASM) cells. Angiotensin II (Ang II) and 5-hydroxytryptamine (5-HT) stimulated both ERK and NHE-1 activities, with activation of NHE-1 preceding that of ERK. The concentration-response curves for 5-HT and Ang II were superimposable for both processes. Inhibition of NHE-1 with pharmacological agents or by isotonic replacement of sodium in the perfusate with choline or tetramethylammonium greatly attenuated ERK activation by 5-HT or Ang II. Similar maneuvers significantly attenuated 5-HT- or Ang II-mediated activation of MEK and Ras but not transphosphorylation of the epidermal growth factor (EGF) receptor. EGF receptor blockade attenuated ERK activation, but not NHE-1 activation by 5-HT and Ang II, suggesting that the EGF receptor and NHE-1 work in parallel to stimulate ERK activity in RASM cells, converging distal to the EGF receptor but at or above the level of Ras in the Ras-MEK-ERK pathway. Receptor-independent activation of NHE-1 by acute acid loading of RASM cells resulted in the rapid phosphorylation of ERK, which could be blocked by pharmacological inhibitors of NHE-1 or by isotonic replacement of sodium, closely linking the proton transport function of NHE-1 to ERK activation. These studies identify NHE as a new regulator of ERK activity in RASM cells.

Collagenase-2 and -3 Mediate Epidermal Growth Factor Receptor Transactivation by Bradykinin B2 Receptor in Kidney Cells

We have previously shown that stimulation of extracellular signal-regulated protein kinase (ERK) by bradykinin (BK) in murine inner medullary collecting duct (mIMCD)-3 cells is mediated by epidermal growth factor receptor (EGFR) transactivation. The mechanism of EGFR transactivation seemed to be novel, because it does not require phospholipase C, Ca2+, calmodulin, protein kinase C, Gαi subunits, or EGFR-B2 receptor heterodimerization. In this study, we demonstrated the involvement of matrix metalloproteinases (MMPs) in B2 receptor-induced EGFR transactivation using their broad-spectrum inhibitors batimastat and N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-l-tryptophan methylamide (Galardin) (GM-6001). Selective inhibitors for collagenase-2 and -3 (MMP-8 and MMP-13, respectively) blocked BK-induced EGFR phosphorylation and ERK activation, whereas inhibitors for MMP-1, -2, -3, -7, or -9 were without effect. Transfection of mIMCD-3 cells with MMP-8 small interfering RNA (siRNA) resulted in ∼50% decrease of BK-induced ERK activation. A neutralizing antibody against MMP-13 as well as transfection with MMP-13 siRNA produced a similar effect. Inhibition of both collagenases resulted in ∼65% decrease of BK-induced ERK activation, supporting roles for both enzymes. Stimulation of mIMCD-3 cells with 10 nM BK increased the activity of collagenases in concentrated culture media within 10 min. Moreover, recombinant MMP-13 and MMP-8, when applied to mIMCD-3 cells for 10 min without BK, stimulated tyrosine phosphorylation of EGFR and caused ∼250% increase over basal ERK phosphorylation comparable with BK-induced ERK activation. Collagenases-induced ERK activation was inhibited by 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG-1478) and thus dependent on EGFR tyrosine kinase activity. This study demonstrates a novel role for collagenase-2 and -3 in signaling of the Gq-coupled BK B2 receptor in mIMCD-3 cells.

Jak2 and Ca2+/calmodulin are key intermediates for bradykinin B-2 receptor-mediated activation of Na+/H+ exchange in KNRK and CHO cells

Na(+)/H(+) exchangers are ubiquitous in mammalian cells, carrying out key functions, such as cell volume defense, acid-base homeostasis, and regulation of the cytoskeleton. We used two screening technologies (FLIPR and microphysiometry) to characterize the signal transduction pathway used by the bradykinin B(2) receptor to activate Na(+)/H(+) exchange in two cell lines, KNRK and CHO. In both cell types, B(2) receptor activation resulted in rapid increases in the rate of proton extrusion that were sodium-dependent and could be blocked by the Na(+)/H(+) exchange inhibitors EIPA and MIA or by replacing extracellular sodium with TMA. Activation of Na(+)/H(+) exchange by bradykinin was concentration-dependent and could be blocked by the selective B(2) receptor antagonist HOE140, but not by the B(1) receptor antagonist des-Arg10-HOE140. Inhibitors of Jak2 tyrosine kinase (genistein and AG490) and of CAM (W-7 and calmidazolium) attenuated bradykinin-induced activation of Na(+)/H(+) exchange. Bradykinin induced formation of a complex between CAM and Jak2, supporting a regulatory role for Jak2 and CAM in the activation of Na(+)/H(+) exchange in KNRK and CHO cells. We propose that this pathway (B(2) receptor --> Jak2 --> CAM --> Na(+)/H(+) exchanger) is a fundamental regulator of Na(+)/H(+) exchange activity.