Inhibition of G protein-coupled receptor signaling by expression of cytoplasmic domains of the receptor.
The third intracellular domain (3i) of G protein-coupled receptors plays a major role in the activation of G proteins. Alterations in this region of the receptor can affect receptor/G protein coupling efficiency and specificity. We recently reported (Luttrell, L. M., Cotecchia, S., Ostrowski, J., Kendall, H., Lefkowitz, R.J. (1993) Science 259, 1453-1457) that coexpression of the 3i domain of the alpha 1B adrenergic receptor (AR) (alpha 1B3i) specifically inhibited alpha 1BAR-mediated inositol phosphate production, with no effect on D1A dopamine receptor (D1ADR)-mediated cAMP production. Similarly, expression of the 3i domain of D1ADR (D1A3i) inhibited D1ADR-mediated cAMP production but did not affect alpha 1BAR-mediated inositol phosphate accumulation. This suggests that peptides derived from a G protein-coupled receptor might serve as antagonists of receptor/G protein interactions. The present studies were performed to test the generality as well as the specificity of this phenomenon. The effect of expression of the second intracellular domain (2i), the 3i domain, and the fourth intracellular domain (4i) of alpha 1BAR on second messenger generation mediated by the alpha 1BAR, the M1 muscarinic cholinergic receptor (M1AChR), and the D1ADR was examined. Although the alpha 1B2i domain had no effect on receptor/G protein coupling for any receptor tested, the alpha 1B3i domain inhibited signaling mediated by alpha 1BAR and M1AChR but not by D1ADR, while the alpha 1B4i domain inhibited signaling mediated by each of the receptors. To investigate the generality of 3i domain-induced inhibition of receptor activity further, the 3i domains of two Gq-coupled receptors (alpha 1BAR and M1AChR) and two Gi-coupled receptors (alpha 2AAR and M2AChR) were tested for effects on the second messenger generation mediated by each of the four receptors. In each case, the homologous 3i domain caused significant inhibition (40-60%), while the 3i domain of the receptor coupled to the same G protein also decreased receptor/G protein coupling. In contrast, receptor/G protein coupling appeared unaffected by expression of 3i domains derived from receptors coupled to different G proteins. The alpha 1B3i domain-provoked inhibition of homologous receptor signaling was surmountable at high receptor density, and assays using a phorbol response element/reporter gene construct detected a weak enhancement of basal second messenger generation in cells expressing the alpha 1B3i domain alone.(ABSTRACT TRUNCATED AT 400 WORDS)
Hawes, BE; Luttrell, LM; Exum, ST; Lefkowitz, RJ
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