RESUMO
BACKGROUND: Calcitonin (CT) exerts an autocrine/paracrine influence on prostatic tumor invasion through coupling to transduction protein Gsalpha. Cell adhesion glycoprotein CD44 variant v7-v10 also faciliates invasion, but its modulation by the CT-Gsalpha system was unexplored. MATERIALS AND METHODS: LnCaP, PC-3 and metastasis-derived PC-3M cell lines were studied, including cells modified therefrom: Gsalpha-QL, expressing mutant constitutively active Gsalpha protein, and CT+, overexpressing CT. CD44 variant expression was evaluated in vivo after orthotopic implantion into nude mice, and in vitro by real-time RT-PCR and Western blotting. RESULTS: Both mRNA and protein levels of the CD44 variant were minimal in PC-3M tumor implants, but elevated in Gsalpha-QL. Exogenous CT stimulated invasion into Matrigel strongly in LnCaP and CT+, and less in PC-3 and Gsalpha-QL. By Western blot analysis, untreated Gsalpha-QL and CT+ cells overexpressed CD44 variant compared with LnCaP or PC-3. By quantitative RT-PCR, exogenous CT dose-dependently increased CD44 variant mRNA to seven-fold. Pharmacologic agents that stimulated or inhibited Gsalpha activity or stimulated adenylyl cyclase produced proportionate dose-dependent effects on both CD44 variant expression and Matrigel invasion. CONCLUSION: This paracrine factor, acting though cyclic AMP, regulates the expression of CD44v7-10, which modulates the tumor phenotype.
Assuntos
Calcitonina/farmacologia , Receptores de Hialuronatos/biossíntese , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Adenilil Ciclases/metabolismo , Animais , Calcitonina/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Subunidades alfa Gs de Proteínas de Ligação ao GTP/biossíntese , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Guanilil Imidodifosfato/farmacologia , Humanos , Receptores de Hialuronatos/genética , Hibridização In Situ , Masculino , Camundongos , Camundongos Nus , Invasividade Neoplásica , Neoplasias da Próstata/genética , Isoformas de Proteínas , RNA Mensageiro/biossíntese , RNA Mensageiro/genéticaRESUMO
Sequence and predicted structural similarities between members of the Cys loop superfamily of ligand-gated ion channel receptors and the acetylcholine binding protein (AChBP) suggest that the ligand-binding site is formed by six loops that intersect at subunit interfaces. We employed site-directed mutagenesis to investigate the role of amino acids from the loop C region of the murine 5-HT(3AS)R in interacting with two structurally different agonists, serotonin (5-HT) and m-chlorophenylbiguanide (mCPBG). Mutant receptors were evaluated using radioligand binding, two-electrode voltage clamp, and immunofluorescence studies. Electrophysiological assays were employed to identify changes in response characteristics and relative efficacies of mCPBG and the partial agonist, 2-methyl 5-HT (2-Me5-HT). We have also constructed novel 5-HT and mCPBG docked models of the receptor binding site based on homology models of the AChBP. Both ligand-docked models correlate well with results from mutagenesis and electrophysiological assays. Four key amino acids were identified as being important to ligand binding and/or gating of the receptor. Among these, I228 and D229 are specific for effects mediated by 5-HT compared to mCPBG, indicating a differential interaction of these ligands with loop C. Residues F226 and Y234 are important for both 5-HT and mCPBG interactions. Mutations at F226, I228, and Y234 also altered the relative efficacies of agonists, suggesting a role in the gating mechanism.