No abstract

RIC‐3 is a transmembrane protein which enhances maturation (folding and assembly) of neuronal nicotinic acetylcholine receptors (nAChRs). In this study, we report the cloning and characterisation of 11 alternatively spliced isoforms of Drosophila melanogaster RIC‐3 (DmRIC‐3). Heterologous expression studies of alternatively spliced DmRIC‐3 isoforms demonstrate that nAChR chaperone activity does not require a predicted coiled‐coil domain which is located entirely within exon 7. In contrast, isoforms containing an additional exon (exon 2), which is located within a proline‐rich N‐terminal region, have a greatly reduced ability to enhance nAChR maturation. The ability of DmRIC‐3 to influence nAChR maturation was examined in co‐expression studies with human α7 nAChRs and with hybrid nAChRs containing both Drosophila and rat nAChR subunits. When expressed in a Drosophila cell line, several of the DmRIC‐3 splice variants enhanced nAChR maturation to a significantly greater extent than observed with human RIC‐3. In contrast, when expressed in a human cell line, human RIC‐3 enhanced nAChR maturation more efficiently than DmRIC‐3. The cloning and characterisation of 11 alternatively spliced DmRIC‐3 isoforms has helped to identify domains influencing RIC‐3 chaperone activity. In addition, studies conducted in different expression systems suggest that additional host cell factors may modulate the chaperone activity of RIC‐3.

The growth factor family of neurotrophins has major roles both inside and outside the nervous system. Here, we report a detailed histological analysis of key phenotypes generated by the ablation of the Kinase D interacting substrate of 220 kDa/Ankyrin repeat-rich membrane spanning (Kidins220/ARMS) protein, a membrane-anchored scaffold for the neurotrophin receptors Trk and p75NTR. Kidins220 is important for heart development, as shown by the severe defects in the outflow tract and ventricle wall formation displayed by the Kidins220 mutant mice. Kidins220 is also important for peripheral nervous system development, as the loss of Kidins220 in vivo caused extensive apoptosis of DRGs and other sensory ganglia. Moreover, the neuronal-specific deletion of this protein leads to early postnatal death, showing that Kidins220 also has a critical function in the postnatal brain.

Kidins220/ARMS is a transmembrane protein playing a crucial role in neuronal and cardiovascular development. Kidins220/ARMS is a downstream target of neurotrophin receptors and interacts with several signalling and trafficking factors. Through computational modelling, we found two potential sites for alternative splicing of Kidins220/ARMS. The first is located between exon 24 and exon 29, while the second site replaces exon 32 by a short alternative terminal exon 33. Here we describe the conserved occurrence of several Kidins220/ARMS splice isoforms at RNA and protein levels. Kidins220/ARMS splice isoforms display spatio-temporal regulation during development with distinct patterns in different neuronal populations. Neurotrophin receptor stimulation in cortical and hippocampal neurons and neuroendocrine cells induces specific Kidins220/ARMS splice isoforms and alters the appearance kinetics of the full-length transcript. Remarkably, alternative terminal exon splicing generates Kidins220/ARMS variants with distinct cellular localisation: Kidins220/ARMS containing exon 32 is targeted to the plasma membrane and neurite tips, whereas Kidins220/ARMS without exon 33 mainly clusters the full-length protein in a perinuclear intracellular compartment in PC12 cells and primary neurons, leading to a change in neurotrophin receptor expression. Overall, this study demonstrates the existence of novel Kidins220/ARMS splice isoforms with unique properties, revealing additional complexity in the functional regulation of neurotrophin receptors, and potentially other signalling pathways involved in neuronal and cardiovascular development.