beta gamma Subunits of pertussis toxin-sensitive G proteins mediate A(1) adenosine receptor agonist-induced activation of phospholipase C in collaboration with thyrotropin - A novel stimulatory mechanism through the cross-talk of two types of receptors

COS-7 cells were transiently transfected with human thyrotropin receptor and dog A(1) adenosine receptor cDNAs. An A(1) agonist, N-6-(L-2-phenylisopropyl) adenosine (PIA), which is ineffective alone, enhanced tale thyrotropin (TSH)-induced inositol phosphate production, reflecting phospholipase C (PLC) activation, but inhibited the TSH-induced cAMP accumulation, reflecting adenylyl cyclase inhibition, These PIA-induced actions were completely inhibited by pertussis toxin (PTX) treatment. Moreover, in the cells expressing a PTX-insensitive mutant of G(i)2 alpha or G(i)3 alpha, in which a glycine residue was substituted for a cysteine residue to be ADP-ribosylated by PTX, at the fourth position of the C terminus, PIA effectively exerted both stimulatory and inhibitory effects on the TSH-induced actions although the cells were treated with the toxin. Overexpression of the beta gamma subunits of the G proteins enhanced the TSH-induced inositol phosphate production without any significant effect on the cAMP response; under these conditions, PIA did not further increase the elevated inositol phosphate response to TSH. On the contrary, overexpression of a constitutively active mutant of G(i)2 alpha, in which the guanosine triphosphatase activity is lost, inhibited the TSH-induced cAMP accumulation brat hardly affected the inositol phosphate response; under these conditions, PIA never exerted further inhibitory effects an the cAMP response to TSH. In contrast to the case of the TSH-induced inositol phosphate response, the response to a constitutively active G(11)alpha mutant was not appreciably affected, and that to NaF was rather inhibited by PIA and overexpression of the beta gamma subunits. Taken together, these results suggest that a single type of PTX-sensitive G protein mediates the A(1) adenosine receptor-linked modulation of two signaling pathways in collaboration with an activated thyrotropin receptor; alpha subunits of the PTX-sensitive G proteins mediate the inhibitory action an adenylyl cyclase, and the beta gamma subunits mediate the stimulatory action on PLC, In the case of the latter stimulatory action on PLC, the beta gamma subunits may not directly activate PLC. The possible mechanism by which beta gamma subunits enhance the TSH-induced PLC activation is discussed.