The guanine nucleotide-binding regulatory protein alpha-subunit, Galpha(16), is primarily expressed in hemopoietic cells, and interacts with a large number of seven-membrane span receptors including chemoattractant receptors. We investigated the biological functions resulting from Galpha(16) coupling of chemoattractant receptors in a transfected cell model system. HeLa cells expressing a kappaB-driven luciferase reporter, Galpha(16), and the formyl peptide receptor responded to fMLP with a approximately 7- to 10-fold increase in luciferase activity. This response was accompanied by phosphorylation of IkappaBalpha and elevation of nuclear kappaB-DNA binding activity, indicating activation of NF-kappaB. In contrast to Galpha(16), expression of Galpha(q), Galpha(13), and Galpha(i2) resulted in a marginal increase in kappaB luciferase activity. A GTPase-deficient, constitutively active Galpha(16) mutant (Q212L) could replace agonist stimulation for activation of NF-kappaB. Furthermore, expression of Galpha(16) (Q212L) markedly enhanced TNF-alpha-induced kappaB reporter activity. The Galpha(16)-mediated NF-kappaB activation was paralleled by an increase in phospholipase C-beta activity, and was blocked by pharmacological inhibitors of protein kinase C (PKC) and by buffering of intracellular Ca(2+). The involvement of a conventional PKC isoform was confirmed by the finding that expression of PKCalpha enhanced the effect of Galpha(16), and a dominant negative PKCalpha partially blocked Galpha(16)-mediated NF-kappaB activation. In addition to formyl peptide receptor, Galpha(16) also enhanced NF-kappaB activation by the C5a and C3a receptors, and by CXC chemokine receptor 2 and CCR8. These results suggest a potential role of Galpha(16) in transcriptional regulation downstream of chemoattractant receptors.