The cysteine-rich with EGF-like domains 2 (CRELD2) protein interacts with the large cytoplasmic domain of human neuronal nicotinic acetylcholine receptor alpha4 and beta2 subunits.

Using a yeast two-hybrid screening we report the isolation of a novel human protein, hCRELD2beta, that interacts specifically with the large cytoplasmic regions of human nicotinic acetylcholine receptor (nAChR) alpha4 and beta2 subunits, both in yeast cells and in vitro. This interaction is not detected with nAChR alpha7 and alpha3 subunits. The hCRELD2 gene encodes for multiple transcripts, likely to produce multiple protein isoforms. A previously reported one has been renamed as CRELD2alpha. Isoforms alpha and beta are expressed in all tissues examined and have the same N-terminal and central regions but alternative C-terminal regions. Both isoforms interact with the alpha4 subunit. Within this subunit the interaction was localized to the N-terminal region of the large cytoplasmic loop. The CRELD2beta protein is present at the endoplasmic reticulum where colocalized with alpha4beta2 nAChRs upon cell transfection. Immunohistochemistry experiments demonstrated the presence of CRELD2 in the rat brain at sites where alpha4beta2 receptors have been previously detected. Labeling was restricted to neuronal perikarya. Finally, CRELD2 decreases the functional expression and impairs membrane transport of alpha4beta2 nAChRs in Xenopus leavis oocytes, without affecting alpha3beta4 and alpha7 nAChR expression. These results suggest that CRELD2 can act as a specific regulator of alpha4beta2 nAChR expression.

Different respiratory control systems are affected in homozygous and heterozygous kreisler mutant mice.

During embryonic development, restricted expression of the regulatory genes Krox20 and kreisler are involved in segmentation and antero-posterior patterning of the hindbrain neural tube. The analysis of transgenic mice in which specific rhombomeres (r) are eliminated points to an important role of segmentation in the generation of neuronal networks controlling vital rhythmic behaviours such as respiration. Thus, elimination of r3 and r5 in Krox20-/- mice suppresses a pontine antiapneic system (Jacquin et al., 1996). We now compare Krox20-/- to kreisler heterozygous (+/kr) and homozygous (kr/kr) mutant neonates. In +/kr mutant mice, we describe hyperactivity of the antiapneic system: analysis of rhythm generation in vitro revealed a pontine modification in keeping with abnormal cell specifications previously reported in r3 (Manzanares et al., 1999b). In kr/kr mice, elimination of r5 abolished all +/kr respiratory traits, suggesting that +/kr hyperactivity of the antiapneic system is mediated through r5-derived territories. Furthermore, collateral chemosensory pathways that normally mediate delayed responses to hypoxia and hyperoxia were not functional in kr/kr mice. We conclude that the pontine antiapneic system originates from r3r4, but not r5. A different rhythm-promoting system originates in r5 and kreisler controls the development of antiapneic and chemosensory signal transmission at this level.