RESUMEN
The secretin receptor is a prototypic class B G protein-coupled receptor that is activated by binding of its natural peptide ligand. The signaling effects of this receptor are mediated by coupling with Gs, which activates cAMP production, and Gq, which activates intracellular calcium mobilization. We have explored the molecular basis for the coupling of each of these G proteins to this receptor using systematic site-directed mutagenesis of key residues within each of the intracellular loop regions, and studying ligand binding and secretin-stimulated cAMP and calcium responses. Mutation of a conserved histidine in the first intracellular loop (H157A and H157R) markedly reduced cell surface expression, resulting in marked reduction in cAMP and elimination of measurable calcium responses. Mutation of an arginine (R153A) in the first intracellular loop reduced calcium, but not cAMP responses. Mutation of a dibasic motif in the second intracellular loop (R231A/K232A) had no significant effects on any measured responses. Mutations in the third intracellular loop involving adjacent lysine and leucine residues (K302A/L303A) or two arginine residues separated by a leucine and an alanine (R318A/R321A) significantly reduced cAMP responses, while the latter also reduced calcium responses. Additive effects were elicited by combining the effective mutations, while combining all the effective mutations resulted in a construct that continued to bind secretin normally, but that elicited no significant cAMP or calcium responses. These data suggest that, while some receptor determinants are clearly shared, there are also distinct determinants for coupling with each of these G proteins.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de la Hormona Gastrointestinal/metabolismo , Secretina/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Calcio/metabolismo , Células Cultivadas , Chlorocebus aethiops , AMP Cíclico/metabolismo , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Unión Proteica , Estructura Terciaria de Proteína , Ratas , Receptores Acoplados a Proteínas G/genética , Receptores de la Hormona Gastrointestinal/genética , Transducción de Señal , TransfecciónRESUMEN
Starvation of Dictyostelium induces a developmental program in which cells form an aggregate that eventually differentiates into a multicellular structure. The aggregate formation is mediated by directional migration of individual cells that quickly transition to group migration in which cells align in a head-to-tail manner to form streams. Cyclic AMP acts as a chemoattractant and its production, secretion, and degradation are highly regulated. A key protein is the extracellular phosphodiesterase PdsA. In this study we examine the role and localization of PdsA during chemotaxis and streaming. We find that pdsA(-) cells respond chemotactically to a narrower range of chemoattractant concentrations compared with wild-type (WT) cells. Moreover, unlike WT cells, pdsA(-) cells do not form streams at low cell densities and form unusual thick and transient streams at high cell densities. We find that the intracellular pool of PdsA is localized to the endoplasmic reticulum, which may provide a compartment for storage and secretion of PdsA. Because we find that cAMP synthesis is normal in cells lacking PdsA, we conclude that signal degradation regulates the external cAMP gradient field generation and that the group migration behavior of these cells is compromised even though their signaling machinery is intact.