Abstract
Punctin/MADD-4, a member of the ADAMTSL extracellular matrix protein family, was identified as an anterograde synaptic organizer in the nematode Caenorhabditis elegans. At GABAergic neuromuscular junctions, the short isoform MADD-4B binds the ectodomain of neuroligin NLG-1, itself a postsynaptic organizer of inhibitory synapses. To identify the molecular bases of their partnership, we generated recombinant forms of the two proteins and carried out a comprehensive biochemical and biophysical study of their interaction, complemented by an in vivo localization study. We show that spontaneous proteolysis of MADD-4B first generates a shorter N-MADD-4B form, which comprises four thrombospondin (TSP) domains and one Ig-like domain and binds NLG-1. A second processing event eliminates the C-terminal Ig-like domain along with the ability of N-MADD-4B to bind NLG-1. These data identify the Ig-like domain as the primary determinant for N-MADD-4B interaction with NLG-1 in vitro We further demonstrate in vivo that this Ig-like domain is essential, albeit not sufficient per se, for efficient recruitment of GABAA receptors at GABAergic synapses in C. elegans The interaction of N-MADD-4B with NLG-1 is also disrupted by heparin, used as a surrogate for the extracellular matrix component, heparan sulfate. High-affinity binding of heparin/heparan sulfate to the Ig-like domain may proceed from surface charge complementarity, as suggested by homology three-dimensional modeling. These data point to N-MADD-4B processing and cell-surface proteoglycan binding as two possible mechanisms to regulate the interaction between MADD-4B and NLG-1 at GABAergic synapses.
Highlights
Punctin/MADD-4, a member of the ADAMTSL extracellular matrix protein family, was identified as an anterograde synaptic organizer in the nematode Caenorhabditis elegans
Besides the NRXs, the extracellular interactome of the NLGs includes secreted partners, such as thrombospondin, whose interaction with NLG accelerates synapse formation [9]; hevin, which is secreted by astrocytes and bridges isoforms NLG1B and NRXa1 at excitatory synapses [10]; and the MAM domain-containing GPI-anchored (MDGA) proteins, which act as negative regulators of synaptic activity by challenging NRX binding to the NLGs [11]
It was proposed that MADD-4L and MADD-4B undergo intermolecular association and that this event inactivates the ability of MADD-4L to recruit NLG-1 and prevents incorrect GABA type A receptor (GABAAR) clustering at cholinergic neuromuscular junction (NMJ) [19, 22]
Summary
Punctin/MADD-4, a member of the ADAMTSL extracellular matrix protein family, was identified as an anterograde synaptic organizer in the nematode Caenorhabditis elegans. These data identify the Ig-like domain as the primary determinant for N-MADD-4B interaction with NLG-1 in vitro. These data point to NMADD-4B processing and cell-surface proteoglycan binding as two possible mechanisms to regulate the interaction between MADD-4B and NLG-1 at GABAergic synapses.
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