β3-adrenoceptor agonist-induced down-regulation of Gsα and functional desensitization in a Chinese hamster ovary cell line expressing a β3-adrenoceptor refractory to down-regulation

Abstract

Chinese hamster ovary (CHO) cells transfected to express human beta 2- or beta 3-adrenoceptors (beta 2-CHO and beta 3-CHO cells) were exposed to the beta-adrenoceptor agonist isoprenaline at various concentrations and for differing times. Sustained exposure of the beta 2-CHO but not beta 3-CHO cells to isoprenaline resulted in a time- and concentration-dependent down-regulation of the receptor as measured by a reduction in specific binding of [125I]cyanopindolol. Such maintained exposure of cells expressing either receptor to the agonist produced a marked down-regulation of immunologically detectable levels of the alpha subunit of the stimulatory guanine-nucleotide-binding protein Gs. This effect was specific for Gs because levels of both G12 alpha and Gq alpha/G11 alpha were unaltered by isoprenaline treatment of both beta 2-CHO and beta 3-CHO cells. The effect of isoprenaline on Gs alpha down-regulation was some 30-fold more potent in the beta 2-CHO than in the beta 3-CHO cells. Time courses of isoprenaline-induced down-regulation of Gs alpha were not different, however, in the two cell lines. Isoprenaline treatment of the beta 3-CHO cells produced a desensitization of agonist-mediated regulation of adenylyl cyclase, manifested by a 4-fold reduction in the potency and a 30% reduction in maximal effect of the agonist, whereas desensitization of the beta 2-CHO cells was considerably greater (25-fold reduction in potency and 70% reduction in maximal effect). These results demonstrate that agonist-induced down-regulation of the G-protein which interacts with a receptor can be produced by both beta 2- and beta 3-adrenoceptors. Despite apparent concurrence of down-regulation of receptors and G-proteins in other systems [e.g. Adie, Mullaney, McKenzie and Milligan (1992) Biochem. J. 285, 529-536], agonist-induced receptor down-regulation does not appear to be a prerequisite for down-regulation of the G-protein. Furthermore, the results suggest that agonist-induced down-regulation of a G-protein may be sufficient, in the absence of receptor regulation, to induce some agonist desensitization of effector function.

The Odd Sibling: Features ofβ3-Adrenoceptor Pharmacology

“…An interesting molecular feature of the b 3 -adrenoceptor is the lack of phosphorylation sites implied in agonist-induced desensitization and downregulation of b 1 -and b 2 -adrenoceptors Nantel et al, 1993). Accordingly, studies with b 3 -adrenoceptors transfected into Chinese hamster ovary cells reported a lack of desensitization with agonist exposure of up to 1 hour (Chaudhry and Granneman, 1994), whereas longer exposure (6-24 hours) resulted in desensitization (Chambers et al, 1994;Candelore et al, 1996); however, the latter occurred in the absence of receptor downregulation and was rather explained by a reduced G s expression (Chambers et al, 1994). On the other hand, b 3 -adrenoceptor desensitization and downregulation can occur when transfected into other cell types such as human embryonic kidney cells (Chaudhry and Granneman, 1994;Michel-Reher and Michel, 2013).…”

(—)‐CGP 12177‐induced increase of human atrial contraction through a putative third β‐adrenoceptor

“…Recent evidence is emerging, however, showing that P3-adrenoceptors can desensitize following a 1 h (Chaudhry & Granneman, 1994) as well as a 24 h exposure to (-)-isoprenaline (Chambers et al, 1994;Nantel et al, 1995) as a function of cell type (Chaudhry & Granneman, 1994;Nantel et al, 1995). Desensitization of f3-adrenoceptors was assessed by a reduction of adenylyl cyclase stimulation (Chaudry & Granneman, 1994;Chambers et al, 1994;Nantel et al, 1995), a reduction of cyclic AMP-dependent protein kinase activity (Nantel et al, 1995) and a reduction of Gs,-protein levels (Chambers et al, 1994).…”

“…Desensitization of f3-adrenoceptors was assessed by a reduction of adenylyl cyclase stimulation (Chaudry & Granneman, 1994;Chambers et al, 1994;Nantel et al, 1995), a reduction of cyclic AMP-dependent protein kinase activity (Nantel et al, 1995) and a reduction of Gs,-protein levels (Chambers et al, 1994). Directly relevant to the fade of the positive inotropic responses to (-)-CGP 12177 is the report of Chaudhry & Granneman (1994) that pretreatment of cells containing transfected human fi3-adrenoceptors with (-)-CGP 12177 also induces reduction of adenylyl cyclase stimulation mediated through these receptors.…”

The tissue distribution of the human β3-adrenoceptor studied using a monoclonal antibody: Direct evidence of the β3-adrenoceptor in human adipose tissue, atrium and skeletal muscle

“…The human b 3 -adrenoceptor gene was obtained under licence from Dr S B Liggett (Duke University Medical Center, Durham, NC), subcloned and transfected into CHO (Chinese Hamster Ovary) dhfr 7 cells as previously described; 24 four subclones expressing different levels of the human b 3 -adrenoceptor were studied, designated C38, C15, D57 and D43 expressing b 3 -adrenoceptors at 130, 400, 1300 and 3000 fmolamg respectively. CHO cells expressing human b 1 -(7000 fmolamg) and b 2 -(2300 fmolamg) adrenoceptors were obtained under licence from A D Strosberg (Universite Paris VII, Institut Cochin de Genetique Moleculaire, Paris) and those expressing the rat b 3 -adrenoceptor (770 fmolamg) from J C Venter (NIH, Bethesda).…”

“…Receptor density values were determined by saturation binding of [ 125 I]-iodocyanopindolol. 24 Immunisation and hybridoma production CBHaCbi rats (National Institute for Medical Research, UK, Mill Hill) were immunised on four occasions with 2Â10 7 CHO b 3 .D43 cells, at 21 ± 28 d intervals intraperitoneally. Three days after the ®nal immunisation, mesenteric lymph node cells were removed and frozen.…”