Regulation of expression of signal transduction cascade elements by G-protein coupled receptors

[摘要] It is now known that external stimuli such as hormones, neurotransmitters, growth factors, light and drugs initiate their biological actions by interacting with receptor macromolecules which are located in the cell membranes. Many transmembrane receptors are coupled to specific effector molecules via G proteins. These trimeric 'receptor-G protein-effector' systems play a primary role in cellular signalling. Molecular cloning techniques have revealed the presence of cDNA species encoding many receptor isoforms or splice variants as well as many G protein subunits. In this study, cDNA species either the long or the short isoforms of the rat TRH receptor were expressed stably in Rat 1 fibroblasts, and clones expressing specific binding of [3H]TRH were detected and expanded. Clones expressing each of these receptors at levels up to 1 pmol/mg of membrane protein were selected for analysis. Reverse transcriptase-PCR on RNA isolated from these clones confirmed that each clone expressed the expected splice variant. Both receptor splice variants bound [3H]TRH with an approximate Kd of 80 nM. In the presence of TRH, both receptor subtypes were able to stimulate inositol phosphate generation in a pertussis toxin-insensitive manner with similar EC50 values. However, despite reports that TRH receptors can also interact with the G proteins Gs and Gi2, neither receptor splice variant was able to modulate adenylyl cyclase activity in either a positive or negative manner. These data indicate that the long and short isoforms of the rat TRH receptor have similar affinities for TRH and display similar abilities to interact with the Gq-like G proteins, but show no ability to regulate adenylyl cyclase in this genetic background. Although downregulation of signal transducer, G protein a subunits, is a well-documented phenomenon the mechanism has been unclear. Maintained exposure of Rat 1 fibroblasts transfected to express the molecularly defined alpha1A/D, alpha1B , and alpha1C adrenergic receptors resulted in a large downregulation of receptors and also a marked downregulation of cellular levels of both of the phosphoinositidase C-linked G proteins, Gqalpha and G11alpha when exposed to the beta1 adrenergic agonist phenylephrine. In order to examine the mechanism of phenylephrine-induced downregulation of Gqalpha and G11alpha, pulse-chase 35S-amino acid labelling experiments were performed with each of the alpha1A/D, alpha1B , and alpha1C adrenergic receptor expressing cell lines. The rate of degradation of Gqalpha and G11alpha, which was adequately modeled by a monoexponential with half-life between 33 and 40h in each of the cell lines in the absence of agonist, was accelerated about 4 fold in the presence of phenylephrine. Each of the alpha1A/D, alpha1B , and alpha1C adrenergic receptor expressing cell lines prelabelled with myo-[3H]inositol were shown to generate high levels of inositol phosphates when exposed to phenylephrine. Additionally, the degree of Gqalpha and G11alpha downregulation in Rat 1 fibroblasts transfected to express either the wild type hamster alpha1B adrenergic receptor or a constitutively active mutant (CAM) form of this receptor was examined. The sustained presence of phenylephrine resulted in substantially greater downregulation in cells expressing the CAMalpha1B adrenergic receptor compared to the wild type at all concentrations of agonist. The enhanced capacity of agonist to stimulate second messenger production at the CAMalpha1B adrenergic receptor and to regulate cellular levels of its associated G proteins by stimulating their rate of degradation is indicative of an enhanced stoichiometry of coupling of this form of the receptor to Gqa and G11alpha Treatment of cells expressing the CAMalpha1B adrenergic receptor with beta1 adrenergic antagonists phentolamine, HV-723, corynanthine, YM-12617, 5-methyl urapidil and WB4101 resulted in upregulation (2-3 fold in 24h) in levels of this receptor. Pretreatment of the CAMalpha1B adrenergic receptor expressing cells with phentolamine caused greater maximal output (about 2-fold) from the Gqalpha and G11alpha to the phosphoinositidase C pathway as well as phospholipase D pathway than the untreated cells upon addition of phenylephrine. However, the potency of phenylephrine was unchanged. By using this system in which the increased receptor levels in response to treatment with phentolamine caused an increase in the basal level of phospholipase D activity, screening of potential inverse agonists was performed. All of the above named ?1 antagonists appeared to be capable of acting as inverse agonists at the CAMalpha1B adrenergic receptor.

[发布机构] University:University of Glasgow

[关键词] Molecular biology