Coexpression of full-length gamma-aminobutyric acid(B) (GABA(B)) receptors with truncated receptors and metabotropic glutamate receptor 4 supports the GABA(B) heterodimer as the functional receptor.
Direct evidence is lacking to show whether the gamma-aminobutyric acid (GABA)(B) gb1-gb2 heterodimer is the signaling form of the receptor. In this study, we tested whether gb1a or gb2 subunits when coexpressed with truncated receptors or metabotropic glutamate receptor mGluR4 could form functional GABA receptors. Coexpression of the ligand binding N-terminal domain of gb1a or the C-terminal portion of gb1a composing the seven-transmembrane segments and intracellular loops with gb2 could not reconstitute functional receptors. We next examined whether mGluR4, which forms homodimers and is structurally related to GABA(B), could act as a surrogate coreceptor for gb1 or gb2. The coexpression of mGluR4 and gb1a led to the expression of gb1a monomers on cell surface membranes as determined by immunoblot analysis and flow cytometry. However, mGluR4-gb1a heterodimers were not formed, and membrane-expressed gb1a monomers were not functionally coupled to adenylyl cyclase in human embryonic kidney 293 cells or activated inwardly rectifying potassium (Kir) channels in Xenopus oocytes. Similarly, the coexpression of mGluR4 and gb2 led to nonfunctional GABA receptors. GABA-activated distal signaling events resulted only after the coexpression and heterodimerization of gb1 and gb2. Taken together with the truncated receptor studies, the data suggest that a high degree of structural specificity is required to form the functional GABA(B) receptor that is a gb1-gb2 heterodimer.
Sullivan
R
R
Department of Biochemistry, Molecular Biology and Chemistry, Merck Frosst Center for Therapeutic Research, Kirkland, Quebec, Canada.
Chateauneuf
A
A
Coulombe
N
N
Kolakowski
L F
LF
Johnson
M P
MP
Hebert
T E
TE
Ethier
N
N
Belley
M
M
Metters
K
K
Abramovitz
M
M
O'Neill
G P
GP
Ng
G Y
GY
D000595:Amino Acid Sequence; D000818:Animals; D015139:Blotting, Southern; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003720:Densitometry; D005434:Flow Cytometry; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D006801:Humans; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D015221:Potassium Channels / Q000378:metabolism; D024661:Potassium Channels, Inwardly Rectifying; D011233:Precipitin Tests; D018080:Receptors, GABA-B / Q000096:biosynthesis* / Q000235:genetics; D018094:Receptors, Metabotropic Glutamate / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014982:Xenopus laevis
D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D008024:Ligands; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D018080:Receptors, GABA-B; D018094:Receptors, Metabotropic Glutamate; D011994:Recombinant Proteins; C108823:metabotropic glutamate receptor 4