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The members of the NF-κB transcription factor family are key regulators of gene expression in the immune response. Different combinations of NF-κB subunits not only diverge in timing to induce transcription but also recognize varying sequences of the NF-κB-binding site of their target genes. The p52 subunit is generated as a result of processing of NF-κB2 p100. Here, we demonstrate that the non-canonical 1κB kinase &3x20AC; (IKK&03F5;) directly interacts with p100. In a transactivation assay, IKK&x03F5; promoted the ability of p52 to transactivate gene expression. This effect was indirect, requiring p65, which was shown to be part of the IKKϵ-p52 complex and to be phosphorylated by IKKϵ, These novel interactions reveal a hitherto unknown function of IKKϵ in the regulation of the alternative NF-κB activation pathway involving p52 and p65. [ABSTRACT FROM AUTHOR]

The γ-aminobutyric acid type B (GABAB) receptor is a heterodimeric G-protein-coupled receptor. In humans, three splice variants of the GABAB receptor 1 (R1) subunit differ in having one, both, or neither of two putative complement control protein (CCP) modules at the extracellular N terminus, prior to the GABA-binding domain. The in vivo function of these predicted modules remains to be discovered, but a likely association with extracellular matrix proteins is intriguing. The portion of the GABAB R1a variant encompassing both of its CCP module-like sequences has been expressed, as have the sequences corresponding to each individual module. Each putative CCP module exhibits the expected pattern of disulfide formation. However, the second module (CCP2) is more compactly folded than the first, and the three-dimensional structure of this more C-terminal module (expressed alone) was solved on the basis of NMRderived nuclear Overhauser effects. This revealed a strong similarity to previously determined CCP module structures in the regulators of complement activation. The N-terminal module (CCP1) displayed conformational heterogeneity under a wide range of conditions whether expressed alone or together with CCP2. Several lines of evidence indicated the presence of native disorder in CCP1, despite the fact that recombinant CCP1 contributes to binding to the extracellular matrix protein fibulin-2. Thus, we have shown that the two CCP modules of GABAB R1a have strikingly different structural properties, reflecting their different functions. [ABSTRACT FROM AUTHOR]

Analysis of interactions between the C-terminal tail of the MOP-1 and MOP-1A variants of the human μ-opioid receptor with proteins derived from a human brain cDNA library resulted in identification of the actin and intermediate filament-binding protein periplakin. Mapping of this interaction indicated that the predicted fourth intracellular loop/helix VIII of the receptor interacts with the C-terminal rod and linker region of periplakin. Periplakin is widely expressed in the central nervous system of both man and rat and demonstrated an overlapping but not identical distribution with μ-opioid (MOP) receptors. Co-expression of periplakin with MOP-1 or a MOP-1-eYFP fusion construct in HEK293 cells did not interfere with agonist-mediated internalization of the receptor. When co-expressed with a MOP1-G[sub i1]α fusion protein periplakin significantly reduced the capacity of the agonist to stimulate binding of [sup 35]S-labeled guanosine 5'-3-O-(thio)triphosphate ([[sup 35]S]GTPγS) to the receptor-associated G protein. By contrast, periplakin did not interfere with agonist-stimulation of [[sup 35]S]GTPγS binding to either an α[sub 2A]-adrenoreceptor-G[sub i1]α fusion protein or a β[sub 2]-adrenoreceptor-G[sub s]α fusion protein, indicating its selectivity of function. This represents the first example of an opioid receptor-interacting protein that functions to disrupt agonist-mediated G protein activation. [ABSTRACT FROM AUTHOR]

Analysis of interactions between the C-terminal tail of the MOP-1 and MOP-1A variants of the human mu-opioid receptor with proteins derived from a human brain cDNA library resulted in identification of the actin and intermediate filament-binding protein periplakin. Mapping of this interaction indicated that the predicted fourth intracellular loop/helix VIII of the receptor interacts with the C-terminal rod and linker region of periplakin. Periplakin is widely expressed in the central nervous system of both man and rat and demonstrated an overlapping but not identical distribution with mu-opioid (MOP) receptors. Co-expression of periplakin with MOP-1 or a MOP-1-eYFP fusion construct in HEK293 cells did not interfere with agonist-mediated internalization of the receptor. When co-expressed with a MOP-1-Gi1 alpha fusion protein periplakin significantly reduced the capacity of the agonist to stimulate binding of 35S-labeled guanosine 5'-3-O-(thio)triphosphate ([35S]GTP gamma S) to the receptor-associated G protein. By contrast, periplakin did not interfere with agonist-stimulation of [35S]GTP gamma S binding to either an alpha 2A-adrenoreceptor-Gi1 alpha fusion protein or a beta2-adrenoreceptor-Gs alpha fusion protein, indicating its selectivity of function. This represents the first example of an opioid receptor-interacting protein that functions to disrupt agonist-mediated G protein activation.