Abstract
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.
Highlights
The somatostatin peptides (SRIF-14 and SRIF-28)1 influence endocrine, exocrine, and neuronal function through binding to a family of six G protein-coupled receptors [1, 2]
Desensitization and Internalization of sst2A Receptor Expressed in GH-R2 Cells—Whereas GH4C1 cells contain low levels (ϳ0.1 pmol/mg of cell protein) of a mixture of the somatostatin receptor subtypes sst1 and sst2 [25, 39]3 the transfected GH-R2 cell line expresses approximately 10 pmol of the sst2A receptor/mg of protein
Desensitization to native SRIF peptides as well as to the clinically used analog SMS or octreotide has been observed in some tissues and cancers but not in others, suggesting that susceptibility to desensitization depends on the sst receptor subtypes present
Summary
The somatostatin peptides (SRIF-14 and SRIF-28) influence endocrine, exocrine, and neuronal function through binding to a family of six G protein-coupled receptors (sst, sst2A, sst2B, sst, sst, and sst5) [1, 2]. Cluding the brain, pituitary, pancreas, spleen, small intestine, and stomach [1, 2], and the receptor protein has recently been shown to be widely distributed in the mammalian brain [3] This receptor isotype mediates many of the central and peripheral actions of SRIF. Exposure to SRIF or to selective SRIF agonists such as SMS 201–995 (SMS) has been reported to lead to desensitization in pituitary cells over the course of hours, days, or weeks [15,16,17,18] Desensitization occurring over both hours [19, 20] and minutes [21] has been reported in the AtT20 corticotropic pituitary cell line, depending on the signaling pathway being examined.
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