The low-density lipoprotein receptor mediates cholesterol homeostasis through endocytosis of lipoproteins. It discharges its ligand in the endosome at pH < 6. In the crystal structure at pH = 5.3, the ligand-binding domain (modules R2 to R7) folds back as an arc over the epidermal growth factor precursor homology domain (the modules A, B, β propeller, and C). The modules R4 and R5, which are critical for lipoprotein binding, associate with the β propeller via their calcium-binding loop. We propose a mechanism for lipoprotein release in the endosome whereby the β propeller functions as an alternate substrate for the ligand-binding domain, binding in a calcium-dependent way and promoting lipoprotein release.

The calcium-independent receptor of α-latrotoxin (CIRL), a neuronal cell surface receptor implicated in the regulation of exocytosis, is a natural chimera of the cell adhesion protein and the G protein-coupled receptor (GPCR). In contrast with canonic GPCRs, CIRL consists of two heterologous non-covalently bound subunits, p120 and p85, due to endogenous proteolytic processing of the receptor precursor in the endoplasmic reticulum. Extracellularly oriented p120 contains hydrophilic cell adhesion domains, whereas p85 resembles a generic GPCR. We determined that the site of the CIRL cleavage is located within a juxtamembrane Cys- and Trp-rich domain of the N-terminal extracellular region of CIRL. Mutations in this domain make CIRL resistant to the cleavage and impair its trafficking. Therefore, we have named it GPS for G protein-coupled receptor proteolysis site. The GPS motif is found in homologous adhesion GPCRs and thus defines a novel receptor family. We postulate that the proteolytic processing and two-subunit structure is a common characteristic feature in the family of GPS-containing adhesion GPCRs.

Poisoning with α-latrotoxin, a neurotoxic protein from black widow spider venom, results in a robust increase of spontaneous synaptic transmission and subsequent degeneration of affected nerve terminals. The neurotoxic action of α-latrotoxin involves extracellular binding to its high affinity receptors as a first step. One of these proteins, CIRL, is a neuronal G-protein-coupled receptor implicated in the regulation of secretion. We now demonstrate that CIRL has two close homologs with a similar domain structure and high degree of overall identity. These novel receptors, which we propose to name CIRL-2 and CIRL-3, together with CIRL (CIRL-1) belong to a recently identified subfamily of large orphan receptors with structural features typical of both G-protein-coupled receptors and cell adhesion proteins. Northern blotting experiments indicate that CIRL-2 is expressed ubiquitously with highest concentrations found in placenta, kidney, spleen, ovary, heart, and lung, whereas CIRL-3 is expressed predominantly in brain similarly to CIRL-1. It appears that CIRL-2 can also bind α-latrotoxin, although its affinity to the toxin is about 14 times less than that of CIRL-1. When overexpressed in chromaffin cells, CIRL-2 increases their sensitivity to α-latrotoxin stimulation but also inhibits Ca²⁺-regulated secretion. Thus, CIRL-2 is a functionally competent receptor of α-latrotoxin. Our findings suggest that although the nervous system is the primary target of low doses of α-latrotoxin, cells of other tissues are also susceptible to the toxic effects of α-latrotoxin because of the presence of CIRL-2, a low affinity receptor of the toxin.

α‐Latrotoxin stimulates neurotransmitter release probably by binding to two receptors, CIRL/latrophilin 1 (CL1) and neurexin Iα. We have now produced recombinant α‐latrotoxin (LtxWT) that is as active as native α‐latrotoxin in triggering synaptic release of glutamate, GABA and norepinephrine. We have also generated three α‐latrotoxin mutants with substitutions in conserved cysteine residues, and a fourth mutant with a four‐residue insertion. All four α‐latrotoxin mutants were found to be unable to trigger release. Interestingly, the insertion mutant LtxN4C exhibited receptor‐binding affinities identical to wild‐type LtxWT, bound to CL1 and neurexin Iα as well as LtxWT, and similarly stimulated synaptic hydrolysis of phosphatidylinositolphosphates. Therefore, receptor binding by α‐latrotoxin and stimulation of phospholipase C are insufficient to trigger exocytosis. This conclusion was confirmed in experiments with La3+ and Cd2+. La3+ blocked release triggered by LtxWT, whereas Cd2+ enhanced it. Both cations, however, had no effect on the stimulation by LtxWT of phosphatidylinositolphosphate hydrolysis. Our data show that receptor binding by α‐latrotoxin and activation of phospholipase C do not by themselves trigger exocytosis. Thus receptors recruit α‐latrotoxin to its point of action without activating exocytosis. Exocytosis probably requires an additional receptor‐independent activity of α‐latrotoxin that is selectively inhibited by the LtxN4C mutation and by La3+.

Receptor-like protein-tyrosine phosphatase sigma (PTPς) is essential for neuronal development and function. Here we report that PTPς is a target of α-latrotoxin, a strong stimulator of neuronal exocytosis. α-Latrotoxin binds to the cell adhesion-like extracellular region of PTPς. This binding results in the stimulation of exocytosis. The toxin-binding site is located in the C-terminal part of the PTPς ectodomain and includes two fibronectin type III repeats. The intracellular catalytic domains of PTPς are not required for the α-latrotoxin binding and secretory response triggered by the toxin in chromaffin cells. These features of PTPς resemble two other previously described α-latrotoxin receptors, neurexin and CIRL. Thus, α-latrotoxin represents an unusual example of the neurotoxin that has three independent, equally potent, and yet structurally distinct targets. The known structural and functional characteristics of PTPς, neurexin, and CIRL suggest that they define a functional family of neuronal membrane receptors with complementary or converging roles in presynaptic function via a mechanism that involves cell-to-cell and cell-to-matrix interaction.

Intraneuronal delivery of a therapeutic, single-domain antibody reverses botulism symptoms and increases survival of mice, guinea pigs, and monkeys.