Asunto(s)
COVID-19/genética , Gravedad del Paciente , Receptores Androgénicos/genética , Anciano , Variación Genética , Humanos , Unidades de Cuidados Intensivos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Admisión del Paciente , Péptidos/genética , Estudios Prospectivos , SARS-CoV-2 , Análisis de Secuencia de ADNRESUMEN
To delineate the role of the cytoplasmic tail in the distinct binding and coupling properties of human dopamine D1-like receptors, chimeric receptors were generated in which the entire tail region of wild-type human D1A (or D1) and D1B (or D5) receptors was exchanged. The hD1A-D1BT, but not hD1B-D1AT, receptor expression was dramatically reduced compared with wild-type receptor expression. Swapping the cytoplasmic tail resulted in a full switch of dopamine binding affinity and constitutive activity, while dopamine potency decreased and agonist-mediated maximal activation of adenylyl cyclase increased for both chimeras. Hence, the cytoplasmic tail plays a crucial role in D1-like receptor expression, agonist binding affinity and constitutive activation but regulates in a distinct fashion the formation of D1A and D1B receptor active states upon dopamine binding.
Asunto(s)
Receptores de Dopamina D1/química , Receptores de Dopamina D1/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Adenilil Ciclasas/metabolismo , Antipsicóticos/metabolismo , Antipsicóticos/farmacología , Sitios de Unión , Línea Celular , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Dopamina/metabolismo , Dopamina/farmacología , Antagonistas de Dopamina/metabolismo , Antagonistas de Dopamina/farmacología , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Expresión Génica , Humanos , Ligandos , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/genética , Receptores de Dopamina D5 , Proteínas Recombinantes de Fusión/agonistas , Proteínas Recombinantes de Fusión/genética , Relación Estructura-Actividad , Termodinámica , TransfecciónRESUMEN
To delineate the structural determinants involved in the constitutive activation of the D1 receptor subtypes, we have constructed chimeras between the D1A and D1B receptors. These chimeras harbored a cognate domain corresponding to transmembrane regions 6 and 7 as well as the third extracellular loop (EL3) and cytoplasmic tail, a domain referred herein to as the terminal receptor locus (TRL). A chimeric D1A receptor harboring the D1B-TRL (chimera 1) displays an increased affinity for dopamine that is indistinguishable from the wild-type D1B receptor. Likewise, a chimeric D1B receptor containing the D1A-TRL cassette (chimera 2) binds dopamine with a reduced affinity that is highly reminiscent of the dopamine affinity for the wild-type D1A receptor. Furthermore, we show that the agonist independent activity of chimera 1 is identical to the wild-type D1B receptor whereas the chimera 2 displays a low agonist independent activity that is indistinguishable from the wild-type D1A receptor. Dopamine potencies for the wild-type D1A and D1B receptor were recapitulated in cells expressing the chimera 2 or chimera 1, respectively. However, the differences observed in agonist-mediated maximal activation of adenylyl cyclase elicited by the D1A and D1B receptors remain unchanged in cells expressing the chimeric receptors. To gain further mechanistic insights into the structural determinants of the TRL involved in the activation properties of the D1 receptor subtypes, we have engineered two additional chimeric D1 receptors that contain the EL3 region of their respective cognate wild-type counterparts (hD1A-EL3B and hD1B-EL3A). In marked contrast to chimera 1 and 2, dopamine affinity and constitutive activation were partially modulated by the exchange of the EL3. Meanwhile, hD1A-EL3B and hD1B-EL3A mutant receptors display a full switch in the agonist-mediated maximal activation, which is reminiscent of their cognate wild-type counterparts. Overall, our studies suggest a fundamental role for the TRL in shaping the intramolecular interactions implicated in the constitutive activation and coupling properties of the dopamine D1 receptor subtypes.
