Characterization of the GABAB1j isoform. A,
schematic representation of the 5′ end of the GABAB1
gene indicating the exons encoding the GABAB1a, GABAB1b,
and GABAB1j isoforms. GABAB1j results from an 870-bp
extension of exon 4 at its 3′ end (exon 4′), generating an open
reading frame of 687 nucleotides encompassing the two SDs. B,
Northern blot analysis of GABAB1a and GABAB1j
transcripts. Total RNA extracted from primary mouse cortical (ctx)
neurons in culture or mouse brain was hybridized to the 32P-labeled
probes indicated in A. The pan probe encodes part of the
extracellular GABA binding domain and detects ∼4.5-kb GABAB1a
and ∼4.1-kb GABAB1b transcripts (not resolved). The SD1/2 probe
encodes the two SDs and detects GABAB1a and ∼1.6-kb
GABAB1j transcripts. The 1j probe encodes 510 nucleotides at the
3′ end of exon 4′. C, in situ hybridization with the
digoxigenin-labeled 1j probe. Top, horizontal section depicting the
dorsal tier of the brain; bottom, high magnification of coronal
section depicting lobules of the cerebellum. The locations of the CA1/3 field
of hippocampus proper (CA1/3), dentate gyrus (DG), medial
habenula (MH), and the granular layer (GL) and molecular
layer (ML) of the cerebellum are indicated. Scale bars, 2 mm
(top) and 200 μm (bottom). D, HEK293 cells
expressing Myc-tagged GABAB1a (myc-1a) or
GABAB1j (myc-1j) proteins. Conditioned medium (cond.
med.) was subjected to immunoprecipitation with a rabbit anti-Myc
antibody and analyzed in parallel with total cell lysate on Western blots
using a mouse anti-Myc antibody. Membrane-bound GABAB1a protein was
selectively detected in the cell lysate, whereas secreted GABAB1j
protein was additionally detected in the cell-conditioned medium. E, left
panel, the anti-SD monoclonal antibody 43H12 immunoprecipitates
GABAB1a but not GABAB1b from mouse brain lysates.
Immunoprecipitated GABAB1 protein (IP anti-SD) was
analyzed in parallel with total brain lysate (input) on Western blots
using a pan GABAB1 antibody
(12). Right panel,
the anti-SD monoclonal antibody 43H12 immunoprecipitates two proteins with a
molecular mass corresponding to that of GABAB1a (**) and
GABAB1j (*) from metabolically labeled cortical neurons.
Radiolabeled proteins were revealed by autoradiography.