1. Neurexins Are Functional α-Latrotoxin Receptors
Shuzo Sugita, Mikhail Khvochtev, Thomas C Südhof 
Neuron 
Volume 22, Issue 3, Pages (March 1999)
DOI: /S (00)

2. Figure 1 Neurexins Function as α-Latrotoxin Receptors
(A) Ca2+-dependent secretion of hGH from PC12 cells transfected with a neurexin 1α expression vector (N1α-1) or a control vector (Control). Transfected PC12 cells were stimulated with the indicated concentrations of recombinant α-latrotoxin in the presence or absence of Ca2+ for 10 min. hGH was measured in the medium and the cells. hGH secretion is expressed as a percentage of total hGH present. Points are from two independent experiments.
(B) Regulation of neurexin 1α-receptor function by alternative splicing. PC12 cells transfected with neurexin 1α without (N1α-1SS4−) or with (N1α-12SS4+) an insert at splice site 4 or with a control vector (Control) were stimulated with low concentrations of α-latrotoxin as indicated. Data are from two independent experiments performed in duplicates. Note expanded α-latrotoxin concentration range compared with (A).
(C) β-Neurexins function as α-latrotoxin receptors regulated by alternative splicing. PC12 cells transfected with neurexin 1β or 2β, with and without inserts in splice site 4 (N1β-1SS4−, N1β-3SS4+, N2β-4SS4−, and N2β-3SS4+, respectively), and control PC12 cells were stimulated with low concentrations of α-latrotoxin. Data are from five independent experiments.
Neuron  , DOI: ( /S (00) )

3. Figure 2
Transfection of Neurexins Does Not Depress the Secretory Response of PC12 Cells to KCl Depolarization
Release of hGH by KCl depolarization was measured in PC12 cells transfected with control plasmid or various neurexin expression vectors. See Experimental Procedures for description of the different neurexin constructs used in the transfections.
Neuron  , DOI: ( /S (00) )

4. Figure 3 Ca2+ Regulation of Exocytosis in PC12 Cells
(A) Ca2+ dependence of hGH secretion in PC12 cells transfected with a control plasmid (Control) or a neurexin 1α expression plasmid (N1α-1SS4−). PC12 cells were stimulated with 1 nM α-latrotoxin for 10 min in medium containing the indicated Ca2+ concentrations. The no Ca2+ condition contained 0.2 mM EGTA.
(B) Concentration dependence of ionomycin-triggered hGH secretion from PC12 cells. PC12 cells were stimulated in the presence of 2.2 mM or 0.0 mM Ca2+ for 10 min with solutions containing the indicated concentrations of ionomycin, and the amount of hGH released was measured as described above. Data are from a single representative experiment and were repeated with separate preparations of ionomycin.
Neuron  , DOI: ( /S (00) )

5. Figure 4
The Intracellular C-Terminal Tail of Neurexin 1α Is Not Required for α-Latrotoxin Receptor Function
Full-length neurexin 1α (N1α-1) or truncated neurexin 1α lacking the last three amino acids (N1α-1Stop1527) or the complete cytoplasmic tail (N1α-1Stop1481) were cotransfected with hGH into PC12 cells. Release was stimulated by three concentrations of α-latrotoxin as shown. Data are from three independent experiments performed in duplicates.
Neuron  , DOI: ( /S (00) )

6. Figure 5
Binding of 125I-Labeled α-Latrotoxin to Immobilized Neurexin–IgG Fusion Proteins: Effects of Alternative Splicing, NaCl, and Ca2+
Binding reactions were performed with IgG fusion proteins containing these extracellular sequences: neurexin 1α without (N1α-1SS4−, [A]) or with (N1α-12SS4+, [B]) an insert in splice site 4; neurexins 1β, 2β, and 3β without an insert in splice site 4 (N1β-1SS4−, N2β-4SS4−, and N3β-1SS4− in [C], [E], and [G], respectively); neurexins 1β and 2β with an insert in splice site 4 (N1β-3SS4+ and N2β-3SS4+ in [D] and [F], respectively); and control protein containing only IgG fused to a signal sequence (H). Equal amounts of IgG proteins (≈2 μg) were immobilized on protein A-Sepharose and incubated with 125I-labeled 0.1 nM α-latrotoxin (12,000 cpm total) before washing in buffered NaCl solutions with the indicated NaCl concentration. Experiments were carried out in the presence and absence of Ca2+ to control for nonspecific binding. Panels show a representative experiment repeated multiple times.
Neuron  , DOI: ( /S (00) )

7. Figure 6
Effect of the Amount of Transfected DNA on the α-Latrotoxin Response in PC12 Cells
PC12 cells were cotransfected with an hGH plasmid and the indicated amounts of a control plasmid or plasmids encoding CL1, neurexin 1α, or neurexin 1β. Transfected cells were stimulated for 10 min with 0.03 nM α-latrotoxin, and the percentage of hGH released was measured. Data shown are from two independent experiments.
Neuron  , DOI: ( /S (00) )

8. Figure 7
Neurexin 1α and CL1 Are Independent, Autonomously Acting α-Latrotoxin Receptors
PC12 cells were transfected individually with neurexin 1α, neurexin 1β, CL1, or control DNA or cotransfected with neurexin 1α and CL1 as indicated. hGH release triggered by various concentrations of α-latrotoxin was measured. Data shown are from three independent experiments.
Neuron  , DOI: ( /S (00) )