Richard B. Clark

Norepinephrine, epinephrine, and histamine cause a rapid increase in the concentration of adenosine 3':5'-cyclic monophosphate (cAMP) in a tumor astrocyte cell line derived from a primary culture of a human glioblastoma multiforme. The catecholamine-induced increase in cAMP is dependent on the cell density, being far greater in cells in the log phase of growth than in cells near terminal density. The response to norepinephrine is inhibited 50% by 0.01 muM propranolol, a blocking agent of beta-adrenergic receptors. In contrast, the effect of histamine on cAMP concentration varies only slightly from log-phase growth to terminal density, and is not inhibited by 10 muM propranolol. The results suggest that astrocytoma cells have independent receptors for catecholamines and histamine. Further, if the astrocytoma cell is an adequate model of the normal glial cell, these results suggest that astrocytes in human cerebral cortex may be sensitive to norepinephrine and histamine.

Previous studies indicated that partial agonists cause less desensitization of the beta2-adrenergic receptor (betaAR) than full agonists; however, the molecular basis for this in intact cells has not been investigated. In the present work, we have determined the rates of desensitization, internalization, and phosphorylation caused by a series of betaAR agonists displaying a 95-fold range of coupling efficiencies. These studies were performed with HEK-293 cells overexpressing the betaAR with hemagglutinin and 6-histidine epitopes introduced into the N and C termini, respectively. This modified betaAR behaved identically to the wild type receptor with regard to agonist Kd, coupling efficiency, and desensitization. The coupling efficiencies for betaAR agonist activation of adenylyl cyclase relative to epinephrine (100%) were 42% for fenoterol, 4.9% for albuterol, 2.5% for dobutamine, and 1.1% for ephedrine. At concentrations of these agonists yielding >90% receptor occupancy, the rate and extent (0-30 min) of agonist-induced desensitization of betaAR activation of adenylyl cyclase followed the same order as coupling efficiency, i.e. epinephrine >/= fenoterol > albuterol > dobutamine > ephedrine. The rate of internalization of the betaAR with respect to these agonists also followed the same order as the desensitization and exhibited a slight lag. Like internalization and desensitization, betaAR phosphorylation exhibited a dependence on agonist strength. The two strongest agonists, epinephrine and fenoterol, provoked 11-13-fold increases in the level of betaAR phosphorylation after just 1 min, whereas the weak agonists dobutamine and ephedrine caused only 3-4-fold increases, similar to levels induced by cAMP-dependent protein kinase activation with forskolin. With longer treatment times, the level of betaAR phosphorylation declined with strong agonists, but it progressively increased with the weaker partial agonists, such that after 30 min the -fold elevation with epinephrine (6.2 +/- 0.82) was not appreciably different from ephedrine (5.0 +/- 0.96) and significantly less than that caused by albuterol (10.4 +/- 1.7). In summary, our results demonstrate an excellent proportionality between the agonist strength and agonist-induced desensitization, internalization, and the rapid initial phase of phosphorylation. The data support the hypothesis that increasing agonist-coupling efficiency primarily affects desensitization by increasing the rate of betaARK phosphorylation of the betaAR.

Cellular responsiveness to the inhibitory peptide somatostatin (SRIF) or its clinically used analogs can desensitize with agonist exposure. While desensitization of other seven-transmembrane domain receptors is mediated by receptor phosphorylation and/or internalization, the mechanisms mediating SRIF receptor (sst) desensitization are unknown. Therefore, we investigated the susceptibility of the sst2A receptor isotype to ligand-induced desensitization, internalization, and phosphorylation in GH-R2 cells, a clone of pituitary tumor cells overexpressing this receptor. A 30-min exposure of cells to either SRIF or the analog SMS 201-995 (SMS) reduced both the potency and efficacy of agonist inhibition of adenylyl cyclase. Internalization of receptor-bound ligand was rapid (t1/2 = 4 min) and temperature-dependent. SRIF and SMS increased the phosphorylation of the 71-kDa sst2A protein 25-fold within 15 min. Receptor phosphorylation was dependent on both the concentration and time of agonist exposure and was not affected by pertussis toxin pretreatment, indicating that receptor occupancy rather than second messenger formation was required. Receptor phosphorylation was also stimulated by phorbol 12-myristate 13-acetate activation of protein kinase C. Both ligand-stimulated and phorbol 12-myristate 13-acetate-stimulated receptor phosphorylation occurred primarily on serine. These studies are the first demonstration of agonist-dependent desensitization, internalization, and phosphorylation of the sst2A receptor and suggest that phosphorylation may mediate the homologous and heterologous regulation of this receptor.