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Getting the Right Receptors to the Synapse

Long and short isoforms of Punctin serve as landmarks for proper clustering of postsynaptic excitatory and inhibitory receptors on muscle cells. Skeletal muscle cells receive both excitatory and inhibitory neuronal inputs. For proper responsiveness, the correct receptors need to cluster at the postsynaptic side across from the excitatory or inhibitory presynaptic side. Pinan-Lucarré et al. identified MADD-4, encoding the homolog of mammalian punctin proteins, in a screen for mutant Caenorhabditis elegans worms that had altered skeletal muscle distribution of L-AChR, the levamisole-sensitive acetylcholine receptor, which responds to excitatory cholinergic input. MADD-4 encodes two long isoforms, MADD-4A and MADD-4C, which are secreted from excitatory motor neurons, and a short isoform, MADD-4B, which only includes the C-terminal part of the longer forms and is secreted from both excitatory and inhibitory motor neurons. Worms genetically null for madd-4 exhibited normal presynaptic morphology, but both the excitatory L-AChR, which respond to cholinergeric motor neurons, and the inhibitory GABAA receptors (GABAARs), which respond to GABAergic motor neurons, were present in clusters in extrasynaptic regions instead of at the appropriate synapses as in the wild-type worms. Ectopic expression of a tagged form of MADD-4A in the muscle cells or in a sensory neuron that innervates a different portion of the muscle resulted in extrasynaptic clustering of L-AChRs that colocalized with ectopic MADD-4A. The introduction of mutations that selectively disrupted production of the short form, but not the long forms, or in madd-4-null worms, transgenic expression of the long forms, but not the short form of MADD-4, restored the localization and abundance of postsynaptic L-AChR and restored L-AChR-dependent evoked currents, indicating that the long forms were necessary and sufficient for L-AChR postsynaptic clustering. In contrast, mutational disruption of production of only the short form resulted in clustering of the GABAARs at the excitatory cholinergic synapse. Furthermore, expression of MADD-4A in the GABAergic motor neurons of madd-4-null worms resulted in clustering of L-AChRs along with the GABAARs at the GABAergic synapse. Expression of both the MADD-4A and MADD-4B in the excitatory motor neurons of madd-4-null worms resulted in fewer GABAARs colocalizing with L-AChRs than expression of MADD-4A in the excitatory motor neurons. The authors proposed that the short form interferes with the GABAAR-clustering activity of the long forms, and coimmunoprecipitation experiments with heterologously expressed proteins confirmed that the various isoforms can interact hetero- and homotypically. Thus, the Punctin family members appear to serve as landmarks for postsynaptic receptor organization. B. Pinan-Lucarré, H. Tu, M. Pierron, P. I. Cruceyra, H. Zhan, C. Stigloher, J. E. Richmond, J.-L. Bessereau, C. elegans Punctin specifies cholinergic versus GABAergic identity of postsynaptic domains. Nature 511, 466–470 (2014). [PubMed]