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(1B)3i) specifically inhibited alpha(1B)AR-mediated inositol phosphate production, with no effect on D-1A dopamine receptor (D(1A)DR)-mediated cAMP production. Similarly, expression of the 3i domain of D(1A)DR (D(1A)3i) inhibited D(1A)DR-mediated cAMP production but did not affect alpha(1B)AR-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(1B)AR on second messenger generation mediated by the alpha(1B)AR, the M(1) muscarinic cholinergic receptor (M(1)AChR), and the D(1A)DR was examined. Although the alpha(1B)2i domain had no effect on receptor/G protein coupling for any receptor tested, the alpha(1B)3i domain inhibited signaling mediated by alpha(1B)AR and M(1)AChR but not by D(1A)DR, while the alpha(1B)4i 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 G(q)-coupled receptors (alpha(1B)AR and M(1)AChR) and two G(i)-coupled receptors (alpha(2A)AR and M(2)AChR) 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(1B)3i 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(1B)3i domain alone. These data demonstrate that disruption of receptor/G protein coupling by 3i domain peptides 1) can be generalized to both G(q)- and G(i)-coupled receptors and 2) Likely results from the ability of these peptides to act as competitive weak partial agonists at the receptor/G protein interface.