Structural Requirements for α-Latrotoxin Binding and α-Latrotoxin-stimulated Secretion: A STUDY WITH CALCIUM-INDEPENDENT RECEPTOR OF α-LATROTOXIN (CIRL) DELETION MUTANTS

Valery Krasnoperov,Mary A Bittner,Ronald W Holz,Oleg Chepurny,Alexander G Petrenko

doi:10.1074/jbc.274.6.3590

Abstract

Stimulation of neurotransmitter release by alpha-latrotoxin requires its binding to the calcium-independent receptor of alpha-latrotoxin (CIRL), an orphan neuronal G protein-coupled receptor. CIRL consists of two noncovalently bound subunits, p85, a heptahelical integral membrane protein, and p120, a large extracellular polypeptide with domains homologous to lectin, olfactomedin, mucin, the secretin receptor family, and a novel structural motif common for large orphan G protein-coupled receptors. The analysis of CIRL deletion mutants indicates that the high affinity alpha-latrotoxin-binding site is located within residues 467-891, which comprise the first transmembrane segment of p85 and the C-terminal half of p120. The N-terminal lectin, olfactomedin, and mucin domains of p120 are not required for the interaction with alpha-latrotoxin. Soluble p120 and all its fragments, which include the 467-770 residues, bind alpha-latrotoxin with low affinity suggesting the importance of membrane-embedded p85 for the stabilization of the complex of the toxin with p120. Two COOH-terminal deletion mutants of CIRL, one with the truncated cytoplasmic domain and the other with only one transmembrane segment left of seven, supported both alpha-latrotoxin-induced calcium uptake in HEK293 cells and alpha-latrotoxin-stimulated secretion when expressed in chromaffin cells, although with a different dose dependence than wild-type CIRL and its N-terminal deletion mutant. Thus the signaling domains of CIRL are not critically important for the stimulation of exocytosis in intact chromaffin cells by alpha-latrotoxin.

Highlights

Stimulation of neurotransmitter release by ␣-latrotoxin requires its binding to the calcium-independent receptor of ␣-latrotoxin (CIRL), an orphan neuronal G protein-coupled receptor
The analysis of CIRL deletion mutants indicates that the high affinity ␣-latrotoxin-binding site is located within residues 467– 891, which comprise the first transmembrane segment of p85 and the C-terminal half of p120
Similar to other G protein-coupled receptors (GPCRs), three intracellular and three extracellular hydrophilic loops in between transmembrane helices can be identified in CIRL

Summary

EXPERIMENTAL PROCEDURES

␣-Latrotoxin was purified from lyophilized black widow spider glands and radioactively labeled with 125I using the chloramine T procedure. N-terminal Deletion Mutants of CIRL—To generate the N-terminal deletion mutants anchored to the membrane-bound fragment of CIRL, AgeI/XbaI-digested pSTR7-6, -7, -8, and -9 plasmids were ligated with a DNA fragment of 3,060 base pairs obtained by digesting pCDR7 with AgeI/XbaI This insert encoded a short COOH-terminal region of p120 and the entire p85 subunit. COS cells were transfected with these plasmids, harvested in 3 days, and analyzed for ␣-latrotoxin binding activity as described below. Purification of the Recombinant Extracellular Domain of CIRL and the Analysis of Its Affinity to ␣-Latrotoxin—COS cells were transfected with an expression plasmid pCDR7N encoding the entire N-terminal extracellular region of CIRL (1– 837 residues) with a His tag at the COOH terminus. Chromaffin cell and HEK293 cell transfections and functional analysis of CIRL mutants were performed as described previously [9, 14]

RESULTS

Because these same soluble CIRL fragments bound quite

DISCUSSION