RESUMEN
G protein-coupled receptors (GPCRs) use a series of conserved microswitches to transmit signals across the cell membrane via an allosteric network encompassing the ligand-binding site and the G protein-binding site. Crystal structures of GPCRs provide snapshots of their inactive and active states, but poorly describe the conformational dynamics of the allosteric network that underlies GPCR activation. Here, we analyzed the correlation between ligand binding and receptor conformation of the α1A-adrenoreceptor, a GPCR that stimulates smooth muscle contraction in response to binding noradrenaline. NMR of [13CϵH3]methionine-labeled α1A-adrenoreceptor variants, each exhibiting differing signaling capacities, revealed how different classes of ligands modulate the conformational equilibria of this receptor. [13CϵH3]Methionine residues near the microswitches exhibited distinct states that correlated with ligand efficacies, supporting a conformational selection mechanism. We propose that allosteric coupling among the microswitches controls the conformation of the α1A-adrenoreceptor and underlies the mechanism of ligand modulation of GPCR signaling in cells.
Asunto(s)
Receptores Adrenérgicos alfa 1/química , Regulación Alostérica , Cristalografía por Rayos X , Humanos , Ligandos , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Receptores Adrenérgicos alfa 1/metabolismoRESUMEN
RESUMEN El CCU es la segunda causa de muerte en mujeres de nuestro país. Dentro de los primeros mecanismos de defensa del hospedero se encuentra la respuesta inmune de las células NK y su función lítica a expensas de su receptor activador NKG2D, el cual posee como ligandos mica, micb y ulbp (1-6), los cuales se expresan en células transformadas y/o infectadas por virus. Uno de los mecanismos de evasión por parte de la célula tumoral es el clivaje de estas proteínas a través de metaloproteinasas como adam10, adam17 y mmp14. Se analizó la expresión de estos ligandos y metaloproteinasas mediante PCR tiempo real, en lineas celulares de referencia para cáncer cervical como HeLa (positiva para VPH-18) y C33A (negativa para VPH). Se obtuvieron valores representativos de expresion relativa genica con diferencias significativas asi: mmp14 en linea HeLa (p= 0.006); y mica y ulbp-3 en la linea C33A (p= 0.020 y p=0.003 respectivamente). Por lo tanto, se podría sugerir que la expresión de mmp14 se encuentra posiblemente involucrados con la presencia de VPH causante del cancer cervical y la respuesta inmunne innata desarrollada.
ABSTRACT Cervical cancer is the second leading cause of death in women in our country. Within the first host defense mechanisms is the immune response of NK cells and their lytic function at the expense of its NKG2D receptor activator which has as ligands mica, micb and ulbp (1-6), which are expressed in transformed cells and / or virally infected. One of the mechanisms of evasion by the tumor cell is the cleavage of these proteins through metalloproteinases as adam10, adam17 and mmp14. We analyzed the expression of these ligands and metalloproteinases by real time PCR, in reference to cell lines HeLa cervical cancer (positive for HPV-18) and C33A (negative for HPV). We obtained representing relative gene expression with significant differences from the other lines of study as follows: mmp14 in HeLa (p = 0.006); and mica and ulbp-3 in C33A (p = 0.020 and p = 0.003 respectively). Thus one might suggest that the expression of mmp14 is possible involved with HPV presence causing high risk of cervical cancer and innate inmunne response developed.