Truncation of the receptor carboxyl terminus impairs agonist-dependent phosphorylation and desensitization of the alpha 1B-adrenergic receptor.

The alpha 1B-adrenergic receptor (alpha 1BAR) and its truncated mutant T368 lacking the last 147 amino acids were stably expressed in Rat1 fibroblasts. The wild type alpha 1BAR was rapidly phosphorylated upon exposure to the agonist epinephrine as well as to phorbol ester as assessed by immunoprecipitation of the receptor with antiserum raised against its amino-terminal portion. Exposure of cells expressing the wild type alpha 1BAR to epinephrine resulted also in rapid homologous desensitization of receptor-mediated response on polyphosphoinositide hydrolysis. On the other hand, truncation of the serine- and threonine-rich carboxyl portion of the alpha 1BAR abolished agonist-induced phosphorylation and greatly impaired homologous desensitization of the receptor. The truncated receptor T368 could undergo agonist-induced decrease of cell surface receptors but to a lesser extent, as compared with the wild type alpha 1BAR. These results demonstrate that the carboxyl portion of the alpha 1BAR plays a crucial role in the regulation of receptor function. They also suggest a strong relationship between agonist-induced phosphorylation and desensitization of the alpha 1BAR, which were both insensitive to the inhibitor of protein kinase C RO-318220. Our findings support the emerging hypothesis that the biochemical mechanisms involved in rapid agonist-dependent regulation of G protein-coupled receptors, which activate polyphosphoinositide hydrolysis, do not primarily involve protein kinase C

Effect of sodium intake on gene expression and plasma levels of ANF in rats.

The effect of short- and long-term sodium loading and sodium restriction on the gene expression as well as on circulating plasma levels of atrial natriuretic factor (ANF) was evaluated in normotensive Wistar rats. These rats were fed either a low-, a regular-, or a high-sodium diet (regular diet and 1% saline as drinking fluid) and studied after 1 and 3 wk. The ANF mRNA was determined in pooled atria and ventricles of the different groups of rats, using the dot-blot technique. Plasma ANF levels were measured with a radioimmunoassay. After 1 wk on the high-sodium diet, ANF mRNA was increased in right atria and ventricles together with circulating ANF levels when compared with animals maintained for the same period on a low-sodium diet. After 3 wk on the various diets, the differences in cardiac ANF mRNA and in plasma ANF levels had disappeared. Gene expression of ANF was also looked for in different areas of the brain, lung, thyroid, adrenals, and the kidney; no hybridization was detected in any of these organs. These data suggest that in rats, the transcription of the ANF gene and peptide release in enhanced only during short-term adaptation to dietary sodium loading

Influence of sodium balance on atrial natriuretic factor in rats with one-kidney, one-clip renal hypertension.

The influence of sodium intake on the gene expression and circulating levels of atrial natriuretic factor (ANF) was investigated in unanesthetized rats with one-kidney, one-clip renal hypertension. After clipping, the rats were maintained for 3 weeks either on a salt-deficient (n = 11) or a regular-sodium diet (n = 10). Animals which had received the regular-sodium diet exhibited significantly higher ANF mRNA levels in their right and left atria than salt-restricted animals, whereas there was no significant difference in plasma ANF levels

Effect of different G protein-coupled receptor kinases on phosphorylation and desensitization of the alpha1B-adrenergic receptor.

The alpha1B-adrenergic receptor (alpha1BAR), its truncated mutant T368, different G protein-coupled receptor kinases (GRK) and arrestin proteins were transiently expressed in COS-7 or HEK293 cells alone and/or in various combinations. Coexpression of beta-adrenergic receptor kinase (betaARK) 1 (GRK2) or 2 (GRK3) could increase epinephrine-induced phosphorylation of the wild type alpha1BAR above basal as compared to that of the receptor expressed alone. On the other hand, overexpression of the dominant negative betaARK (K220R) mutant impaired agonist-induced phosphorylation of the receptor. Overexpression of GRK6 could also increase epinephrine-induced phosphorylation of the receptor, whereas GRK5 enhanced basal but not agonist-induced phosphorylation of the alpha1BAR. Increasing coexpression of betaARK1 or betaARK2 resulted in the progressive attenuation of the alpha1BAR-mediated response on polyphosphoinositide (PI) hydrolysis. However, coexpression of betaARK1 or 2 at low levels did not significantly impair the PI response mediated by the truncated alpha1BAR mutant T368, lacking the C terminus, which is involved in agonist-induced desensitization and phosphorylation of the receptor. Similar attenuation of the receptor-mediated PI response was also observed for the wild type alpha1BAR, but not for its truncated mutant, when the receptor was coexpressed with beta-arrestin 1 or beta-arrestin 2. Despite their pronounced effect on phosphorylation of the alpha1BAR, overexpression of GRK5 or GRK6 did not affect the receptor-mediated response. In conclusion, our results provide the first evidence that betaARK1 and 2 as well as arrestin proteins might be involved in agonist-induced regulation of the alpha1BAR. They also identify the alpha1BAR as a potential phosphorylation substrate of GRK5 and GRK6. However, the physiological implications of GRK5- and GRK6-mediated phosphorylation of the alpha1BAR remain to be elucidated