Abstract
The nature of melatonin receptor-Gi-protein coupling in ovine pars tuberalis (PT) cells of the pituitary was addressed using cholera (CTX) and pertussis (PTX) toxins. ADP-ribosylation of ovine PT membrane proteins using P-32-NAD in the presence of CTX radiolabelled several substrates including 44, 51, and 60 kD proteins. Each were clearly distinct from the 40 kD substrate radiolabelled in the presence of PTX, Acute incubation of PT membranes with either toxin reduced the number of high affinity binding sites for I-125-MEL, although the magnitude of the inhibition was much greater for CTX (56%) than for PTX (20%), A CTX-sensitive component also mediates the inhibition of forskolin-stimulated cyclic AMP accumulation as pre-treatment of PT cells with CTX (5 mu g/ml) for 16 h blocked this response,
G(s) alpha is a major substrate for ADP-ribosylation by CTX, and 16 h pre-treatment of PT cells with CTX (5 mu g/ml) caused a down-regulation of G(s) alpha. Northern analysis showed only one major transcript of G(s) alpha of about 2 kb, which would encompass all of the known splice variants of the G(s) gene. Screening of a cDNA library from ovine PT for G(s)-related genes and sequencing of clones, combined with RT-PCR of PT mRNA, revealed no novel products. On this basis it is concluded that the CTX substrate is unlikely to be a novel splice variant or related gene product of the G(s) class of G-protein. These results indicate that a distinct CTX-sensitive mechanism mediates the inhibition of cyclic AMP by the melatonin receptor, which involves either a novel alpha-sub-unit of a heterotrimeric G-protein or a protein which associates and functionally modulates the activity of the melatonin receptor/G-protein complex.
Original language | English |
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Pages (from-to) | 361-369 |
Number of pages | 9 |
Journal | Journal of Neuroendocrinology |
Volume | 7 |
Issue number | 5 |
DOIs | |
Publication status | Published - May 1995 |
Keywords
- G-protein
- receptor
- cholera toxin
- pars tuberalis
- signal transduction protein
- pertussis toxin
- adenylate-cyclase
- alpha-subunit
- ADP-ribosylation
- rat pituitary
- binding-proteins
- GS
- cells