Constraints on proper folding of the amino terminal domains of group III metabotropic glutamate receptors.
The glutamate binding site of the G-protein coupled metabotropic glutamate receptors (mGluRs) is contained within the large extracellular amino terminal domain (ATD) of the receptor. In this study, we examined the ligand binding properties and cellular dispositions of the membrane-bound mGluR4 and mGluR8 subtypes of mGluRs, and a series of truncated versions of these receptors. Truncation of the ATDs of mGluR4 and mGluR8 40 amino acids upstream of the first transmembrane domain produced soluble proteins that were secreted into the cell culture media of transfected human embryonic kidney cells. The soluble receptors retained ligand binding capabilities. Additional constructs of the ATDs of mGluR4 and mGluR8 were assessed for their ability to bind the agonist [(3)H]L-AP4 and for secretion from cells. A shorter mGluR4 construct truncated 98 amino acids upstream from the first transmembrane domain failed to bind [(3)H]L-AP4, while the analogous mGluR8 construct displayed a low level of binding. Unlike the full-length receptors, which were expressed on the cell surface, or the soluble constructs which were secreted, the shorter constructs were primarily associated with intracellular membranes. These observations suggest that the cysteine-rich region may be important for efficient secretion, but not absolutely obligatory for ligand binding. Surprisingly, longer constructs encoding the entire ATDs of mGluR4 and mGluR8 failed to bind ligand and were localized intracellularly. Together, these findings demonstrate that there are strict limitations on the proper folding of truncated versions of the ATDs of mGluR4 and mGluR8. Specifically, all of the leucine-isoleucine-valine binding protein homology region, and part of the cysteine-rich region is required for optimal secretion in a soluble form that retains ligand binding activity.
Peltekova
V
V
Faculty of Pharmacy and Department of Pharmacology, University of Toronto, 19 Russell Street, Toronto, ON, Canada.
Han
G
G
Soleymanlou
N
N
Hampson
D R
DR
D000818:Animals; D002460:Cell Line; D006801:Humans; D015151:Immunoblotting; D008024:Ligands; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011869:Radioligand Assay; D051381:Rats; D018094:Receptors, Metabotropic Glutamate / Q000378:metabolism*; D012995:Solubility; D014162:Transfection
D008024:Ligands; D018094:Receptors, Metabotropic Glutamate; C111435:metabotropic glutamate receptor 8; C108823:metabotropic glutamate receptor 4