The N-terminal domain of human GABA receptor rho1 subunits contains signals for homooligomeric and heterooligomeric interaction.
gamma-Aminobutyric acid type C (GABAC) receptors identified in retina appear to be composed of GABA rho subunits. The purpose of this study was to localize signals for homooligomeric assembly of rho1 subunits and to investigate whether the same region contained signals for heterooligomeric interaction with rho2 subunits. In vitro translated human rho1 was shown to be membrane-associated, and proteinase K susceptibility studies indicated that the N terminus was oriented in the lumen of ER-derived microsomal vesicles. This orientation suggested the involvement of the N terminus of rho1 in the initial steps of subunit assembly. To test this hypothesis, mutants were created containing only N-terminal sequences (N-rho1) or C-terminal sequences (C-rho1) of rho1. Co-immunoprecipitation studies revealed that N-rho1, but not C-rho1, interacted with rho1 in vitro. When coexpressed in Xenopus oocytes, N-rho1 interfered with rho1 receptor formation. Together, these data suggested that signals for rho1 homooligomeric assembly reside in the N-terminal half of the subunit. Sequential immunoprecipitations were then performed upon cotranslated rho1 and rho2 subunits which demonstrated that rho1 and rho2 interacted in vitro. Co-immunoprecipitation indicated that N-rho1 specifically associated with rho2. Therefore, the N-terminal regions of rho subunits contain the initial signals for both homooligomeric and heterooligomeric assembly into receptors with GABAC properties.
Hackam
A S
AS
Center for Medical Genetics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, USA.
Wang
T L
TL
Guggino
W B
WB
Cutting
G R
GR
D000818:Animals; D000906:Antibodies / Q000276:immunology; D019286:Endopeptidase K / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008861:Microsomes / Q000378:metabolism; D008958:Models, Molecular; D014176:Protein Biosynthesis; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000737:chemistry / Q000378:metabolism*; D018079:Receptors, GABA / Q000737:chemistry / Q000378:metabolism*; D011963:Receptors, GABA-A; D018080:Receptors, GABA-B; D014981:Xenopus
D000906:Antibodies; C087200:GABA-C receptor; C106350:GABRR2 protein, human; D021382:Protein Sorting Signals; D018079:Receptors, GABA; D011963:Receptors, GABA-A; D018080:Receptors, GABA-B; D019286:Endopeptidase K