Biomedical subjects

M Margeta-Mitrovic

Publications and source records attributed to M Margeta-Mitrovic.

5 recordsLinked to original sources

PubMed2001

Function of GB1 and GB2 subunits in G protein coupling of GABA(B) receptors.

Many G protein-coupled receptors (GPCRs) have recently been shown to dimerize, and it was suggested that dimerization may be a prerequisite for G protein coupling. gamma-aminobutyric acid type B (GABA(B)) receptors (GPCRs for GABA, a major inhibitory neurotransmitter in the brain) are obligate heterodimers of homologous GB1 and GB2 subunits, neither of which is functional on its own. This feature of GABA(B) receptors allowed us to examine which of the eight intracellular segments of the heterodimeric receptor were important for G protein activation. Replacing any of the three intracellular loops of GB2 with their GB1 counterparts resulted in nonfunctional receptors. The deletion of the complete GB2 C terminus significantly attenuated the receptor function; however, the proximal 36 residues were sufficient for reconstitution of wild type-like receptor activity. In contrast, the GB1 C terminus could be deleted and GB1 intracellular loops replaced with their GB2 or mGluR1 equivalents without affecting the receptor function. In addition, a large portion of the GB1 i2 loop could be replaced with a random coil peptide without any functional consequences. Thus, GB2 intracellular segments are solely responsible for specific coupling of GABA(B) receptors to their physiologic effectors, G(i) and G protein-activated K(+) channels. These findings strongly support a model in which a single GPCR monomer is sufficient for all of the specific G protein contacts.

Amino Acid Sequence↗

PubMed2001

Ligand-induced signal transduction within heterodimeric GABA(B) receptor.

gamma-aminobutyric acid type B (GABA(B)) receptors, G protein-coupled receptors (GPCRs) for GABA, are obligate heterodimers of two homologous subunits, GB1 and GB2. Typical for family C GPCRs, the N termini of both GB1 and GB2 contain a domain with homology to bacterial periplasmic amino acid-binding proteins (PBPs), but only the GB1 PBP-like domain binds GABA. We found that both GB1 and GB2 extracellular N termini are required for normal coupling of GABA(B) receptors to their physiological effectors, G(i) and G protein-activated K(+) channels (GIRKs). Receptors with two GB2 N termini did not respond to GABA, whereas receptors with two GB1 N termini showed increased basal activity and responded to GABA with inhibition, rather than activation, of GIRK channels. This GABA-induced GIRK current inhibition depended on GABA binding to the chimeric GB(1/2) subunit (the GB1 N-terminal domain attached to the heptahelical domain of GB2), rather than the wild-type GB1 subunit. Interestingly, receptors with reciprocal exchange of N-terminal domains between the subunits were functionally indistinguishable from wild-type receptors. We also found that peptide linkers between GB1 and GB2 PBP-like domains and respective heptahelical domains could be altered without affecting receptor function. This finding suggests that other contacts between the PBP-like and heptahelical domains underlie ligand-induced signal transduction, a finding likely to be relevant for all family C GPCRs.

Amino Acid Sequence↗

PubMed2000

A trafficking checkpoint controls GABA(B) receptor heterodimerization.

Surface expression of GABA(B) receptors requires heterodimerization of GB1 and GB2 subunits, but little is known about mechanisms that ensure efficient heterodimer assembly. We found that expression of the GB1 subunit on the cell surface is prevented through a C-terminal retention motif RXR(R); this sequence is reminiscent of the ER retention/retrieval motif RKR identified in subunits of the ATP-sensitive K+ channel. Interaction of GB1 and GB2 through their C-terminal coiled-coil alpha helices masks the retention signal in GB1, allowing the plasma membrane expression of the assembled complexes. Because individual GABA(B) receptor subunits and improperly assembled receptor complexes are not functional even if expressed on the cell surface, we conclude that a trafficking checkpoint ensures efficient assembly of functional GABA(B) receptors.

Amino Acid Sequence↗

PubMed1999

Immunohistochemical localization of GABA(B) receptors in the rat central nervous system.

The recent cloning of two gamma-aminobutyric acid(B) (GABA(B)) receptor isoforms (GABA(B)R1a/b), which are probably splice variants of the same gene transcript, allowed us to develop an antiserum that recognized the receptors in fixed tissue and to map their distribution in the rat central nervous system (CNS). We also investigated whether GABA(B)R1 colocalizes with glutamic acid decarboxylase (GAD), a marker of GABAergic cell bodies and terminals. Although GABA(B)R1-like immunoreactivity (GABA(B)R1-LI) was distributed throughout the CNS, several distinct distribution patterns emerged: (1) all monoaminergic brainstem cell groups appeared to contain very high levels of GABA(B)R1, (2) a very high intensity of GABA(B)R1-LI was observed in the majority of the cholinergic regions in the CNS, with exception of motoneurons of the third through sixth cranial nerve nuclei, and (3) a low density of the receptor was observed in most of the nuclei that contain cell bodies of GABAergic projection neurons. The highest GABA(B)R1 labeling was observed in the thalamus, interpeduncular nucleus and medial habenula. Cell bodies were labeled throughout the neuroaxis. We also observed dense neuropil labeling in many regions, suggesting that this receptor is localized in dendrites and/or axon terminals. However, in immunofluorescent double-labeling experiments for GABA(B)R1 and GAD, we never observed GABA(B)R1-LI in GAD-positive axon terminals; this result suggests that the GABA(B)R1 may not function as an autoreceptor. Double labeling was observed in the cell bodies of Purkinje neurons and in some interneurons. In general, the immunohistochemical localization of the GABA(B)R1 correlates well with physiologic and autoradiographic data on the distribution of GABA(B) receptors, but some critical differences were noted. Thus, it is likely that additional GABA(B) receptor subtypes remain to be identified.

Animals↗

PubMed1997

Neurotensin and substance P inhibit low- and high-voltage-activated Ca2+ channels in cultured newborn rat nucleus basalis neurons.

Inhibition of Ca2+ currents by the excitatory neurotransmitters neurotensin and substance P was investigated in cultured nucleus basalis neurons with the use of the whole cell patch-clamp technique. The whole cell Ca2+ current, elicited from a holding potential of -80 mV by a step pulse to 0 mV and measured at 100 ms, was inhibited 67.9% by neurotensin and 57.6% by substance P. Low-voltage-activated (LVA) Ca2+ current, elicited by a step pulse to -40 mV from a holding potential of -90 mV, was inhibited by both neurotensin (26.2%) and substance P (24.1%). High-voltage-activated Ca2+ currents were separated with the use of the Ca2+ channel antagonists. Nimodipine (3 microM) inhibited 24.2% of the whole cell Ca2+ current elicited by a step to 0 or +10 mV and measured at 100 ms. Under the same conditions, omega-conotoxin (omega-CgTx)-GVIA (0.5 microM) inhibited 46.4%, omega-CgTx-GVIA + nimodipine 58.7%, and omega-CgTx-MVIIC (5 microM) + nimodipine 75.7% of the current. Omega-Agatoxin (omega-Aga)-IVA (100 nM) did not produce any effect. Neurotensin inhibition of the whole cell Ca2+ current was attenuated by each of these treatments except for the omega-Aga-IVA treatment, which did not change the neurotensin effect. In contrast, neither the omega-Aga-IVA nor the nimodipine treatment had any effect on the substance-P-induced inhibition; the rest of the treatments attenuated the substance-P-induced response. Thus the data indicate that nucleus basalis neurons express LVA as well as L-, N-, and Q-type, but not the P-type, Ca2+ currents. N- and Q-type HVA Ca2+ currents, as well as LVA Ca2+ currents, are inhibited by both neurotensin and substance P. In contrast, L-type current is inhibited by neurotensin but not by substance P. In addition, a fraction of the total whole cell current was resistant to all Ca2+ channel antagonists and thus may correspond to the R-type Ca2+ current. This residual current was inhibited by both neurotensin and substance P. The inhibition of the whole cell Ca2+ current produced by both neurotransmitters was voltage independent, because a large depolarization (+70 mV) was not able to relieve either effect. In cells loaded with 0.1 mM guanosine 5'-[gamma-thio]triphosphate, response to both neurotensin and substance P became irreversible, indicating that the effects of both neurotransmitters were mediated through G proteins. However, pertussis toxin did not affect either the neurotensin or the substance P response.

Animals↗