Distinct Guanine Nucleotide Binding Protein Alpha-Subunit Receptor Coupling in GH Cell Lines: <i>Effects of Bromocriptine and Hormones on Effector Enzyme Modulation</i>

Abstract

The purpose of the present study was to elucidate how the dopamine agonist bromocriptine affected receptor-effector systems in GH cells by measuring adenylate cyclase (AC) and phospholipase C (PL-C) modulation in cell membrane preparations. To perturb the interaction between the receptor and G-protein, polyclonal antibodies reacting with the predicted C-terminal amino acid sequence of G-protein α-subunits were used. The effect of bromocriptine on secretagogue elicited prolactin (PRL) secretion from whole cells was also monitored. Bromocriptine inhibited the basal secretion of PRL in a dose dependent manner, and completely abolished both the thyroliberin (TRH) and the vasoactive intestinal peptide (VIP) stimulated PRL secretion in GH₃ cells. Maximal inhibitory effect on PRL egress elicited by both hormones was obtained at 10-50 µM of bromocriptine. Messenger RNAs for both the short and long form of the D₂ receptor (D₂R) were demonstrated in all three GH cell lines using the RT-PCR technique, advocating that D₂Rs are coupled to distinct G-proteins and, thus, probably being responsible for the observed effects of bromocriptine in these cell lines. Basal AC activity, as measured in membrane preparations of GH₃ cells, remained unaffected by bromocriptine treatment (10 µM), while TRH and VIP stimulated AC activities (175% and 350% of control values, respectively) were partially inhibited (by some 50%). This inhibitory effect of bromocriptine was completely and specifically abolished in the presence of an antiserum against G_i2α. Basal PL-C activity was also unaffected by bromocriptine, while TRH stimulated PL-C activity (350% of control value) was inhibited by bromocriptine (10 µM) by approximately 50%. Immunoblocking of G_q/11α, however, reduced the stimulatory effect of TRH on PL-C activation by some 65%, while an antiserum against G_oα partly counteracted the inhibitory effect of bromocriptine (10 µM) on TRH stimulated PL-C activity. Thus, TRH dependent AC stimulation was counteracted by bromocriptine via G_i2. TRH activation of PL-C occurs via G_q/11, while inhibition by bromocriptine appears to involve G_o. These mechanisms probably account for the major part of the actions of bromocriptine, however, other not yet recognised intermediates may be involved.

Mammalian G Proteins and Their Cell Type Specific Functions

“…With respect to pituitary function, patients with inactivating GNAS mutations show variable degrees of GHRH resistance (415), GH deficiency (210), or hypoprolactinemia (88). In accordance with the important role of G s family G proteins in lactotrophs and somatotrophs, hypothalamic inhibiting hormones, like dopamine or somatostatin, act through G i -coupled receptors (311,536).…”

Thyrotrophin-releasing hormone receptor 1 and prothyrotrophin-releasing hormone mRNA expression in the central nervous system are regulated by suckling in lactating rats

“…Bromocriptine acts on dopamine D2 receptors which are also present in the pituitary (43). The detailed molecular actions of bromocriptine (2-bromo-alphaergocryptine), a semi-synthetic ergot alkaloid with long-term stimulatory effects on dopamine D2 receptors, are not known.…”

Biochemical and physiological insights into TRH receptor-mediated signaling

“…Besides that, Gβγ dimers also contribute to the regulation of various effectors. Previous studies have shown that TRH-R is quite promiscuous, coupling predominantly to G q/11 but also interacting with G s and G i proteins in GH3 cells ( Paulssen et al, 1992a ; Johansen et al, 2001b ; Boutin et al, 2012 ). Binding of a ligand to TRH-R results in an exchange of GDP for GTP at the Gα subunit.…”

“…Nevertheless, TRH-R turned out to be able to activate more than one second messenger system under certain circumstances. In addition to the G q/11 -regulated PLC pathway, TRH-R may also activate AC through direct interaction with the stimulatory G s protein ( Paulssen et al, 1992a ; Johansen et al, 2001b ). In addition, there is some evidence from experiments with GH3 cells that TRH-R can couple to the inhibitory G i proteins that do not cause activation of AC ( Paulssen et al, 1992a ).…”