RESUMEN
ß-adrenergic receptors (ßARs) are critical regulators of acute cardiovascular physiology. In response to elevated catecholamine stimulation during development of congestive heart failure (CHF), chronic activation of Gs-dependent ß1AR and Gi-dependent ß2AR pathways leads to enhanced cardiomyocyte death, reduced ß1AR expression, and decreased inotropic reserve. ß-blockers act to block excessive catecholamine stimulation of ßARs to decrease cellular apoptotic signaling and normalize ß1AR expression and inotropy. Whereas these actions reduce cardiac remodeling and mortality outcomes, the effects are not sustained. Converse to G-protein-dependent signaling, ß-arrestin-dependent signaling promotes cardiomyocyte survival. Given that ß2AR expression is unaltered in CHF, a ß-arrestin-biased agonist that operates through the ß2AR represents a potentially useful therapeutic approach. Carvedilol, a currently prescribed nonselective ß-blocker, has been classified as a ß-arrestin-biased agonist that can inhibit basal signaling from ßARs and also stimulate cell survival signaling pathways. To understand the relative contribution of ß-arrestin bias to the efficacy of select ß-blockers, a specific ß-arrestin-biased pepducin for the ß2AR, intracellular loop (ICL)1-9, was used to decouple ß-arrestin-biased signaling from occupation of the orthosteric ligand-binding pocket. With similar efficacy to carvedilol, ICL1-9 was able to promote ß2AR phosphorylation, ß-arrestin recruitment, ß2AR internalization, and ß-arrestin-biased signaling. Interestingly, ICL1-9 was also able to induce ß2AR- and ß-arrestin-dependent and Ca(2+)-independent contractility in primary adult murine cardiomyocytes, whereas carvedilol had no efficacy. Thus, ICL1-9 is an effective tool to access a pharmacological profile stimulating cardioprotective signaling and inotropic effects through the ß2AR and serves as a model for the next generation of cardiovascular drug development.
Asunto(s)
Antagonistas Adrenérgicos beta/farmacología , Carbazoles/farmacología , Insuficiencia Cardíaca/tratamiento farmacológico , Lipopéptidos/farmacología , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Propanolaminas/farmacología , Antagonistas Adrenérgicos beta/uso terapéutico , Animales , Carbazoles/uso terapéutico , Carvedilol , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , Células HEK293 , Humanos , Lipopéptidos/uso terapéutico , Ratones , Cultivo Primario de Células , Propanolaminas/uso terapéutico , Conformación Proteica/efectos de los fármacos , beta-Arrestinas/agonistasRESUMEN
Here we demonstrate that phosphorylation of the sphingosine-1-phosphate (S1P) receptor S1P(3) is increased specifically in response to S1P. Truncation of the receptor's carboxyl-terminal domain revealed that the presence of a serine-rich stretch of residues between Leu332 and Val352 was essential to observe this effect. Although agonist-occupied wild-type (WT) S1P(3) could be phosphorylated in vitro by G-protein-coupled receptor kinase 2 (GRK2), a role of S1P(3) phosphorylation in controlling S1P(3)-G(q/11) coupling was excluded since A) a phosphorylation-resistant S1P(3) mutant desensitised in a manner indistinguishable from the WT receptor and was phosphorylated to a greater extent than the WT receptor by GRK2 in vitro, and B) co-expression with GRK2 or GRK3 failed to potentiate S1P(3) phosphorylation. S1P(3) phosphorylation was also not required for receptor sequestration away from the cell surface. Together, these data suggest that S1P(3) function is not subject to conventional regulation by GRK phosphorylation and that novel aspects of S1P(3) function distinct from classical G-protein coupling and receptor internalisation may be controlled its carboxyl-terminal domain.