GABAB receptors assemble from GABAB1 and GABAB2 subunits. GABAB2 additionally associates with auxiliary KCTD subunits (named after their K(+) channel tetramerization-domain). GABAB receptors couple to heterotrimeric G-proteins and activate inwardly-rectifying K(+) channels through the βγ subunits released from the G-protein. Receptor-activated K(+) currents desensitize in the sustained presence of agonist to avoid excessive effects on neuronal activity. Desensitization of K(+) currents integrates distinct mechanistic underpinnings. GABAB receptor activity reduces protein kinase-A activity, which reduces phosphorylation of serine-892 in GABAB2 and promotes receptor degradation. This form of desensitization operates on the time scale of several minutes to hours. A faster form of desensitization is induced by the auxiliary subunit KCTD12, which interferes with channel activation by binding to the G-protein βγ subunits. Here we show that the two mechanisms of desensitization influence each other. Serine-892 phosphorylation in heterologous cells rearranges KCTD12 at the receptor and slows KCTD12-induced desensitization. Likewise, protein kinase-A activation in hippocampal neurons slows fast desensitization of GABAB receptor-activated K(+) currents while protein kinase-A inhibition accelerates fast desensitization. Protein kinase-A fails to regulate fast desensitization in KCTD12 knock-out mice or knock-in mice with a serine-892 to alanine mutation, thus demonstrating that serine-892 phosphorylation regulates KCTD12-induced desensitization in vivo. Fast current desensitization is accelerated in hippocampal neurons carrying the serine-892 to alanine mutation, showing that tonic serine-892 phosphorylation normally limits KCTD12-induced desensitization. Tonic serine-892 phosphorylation is in turn promoted by assembly of receptors with KCTD12. This cross-regulation of serine-892 phosphorylation and KCTD12 activity sharpens the response during repeated receptor activation.

• Klíčová slova
• G-protein coupled receptor, GABA-B, GPCR, Kir3, PKA,
• MeSH
• alanin genetika   metabolismus   MeSH
• CHO buňky MeSH
• Cricetulus MeSH
• draslík metabolismus   MeSH
• fosforylace MeSH
• hipokampus cytologie   metabolismus   MeSH
• kultivované buňky MeSH
• metoda terčíkového zámku MeSH
• myši knockoutované MeSH
• myši MeSH
• neurony metabolismus   MeSH
• proteinkinasy závislé na cyklickém AMP metabolismus   MeSH
• proteiny vázající GTP metabolismus   MeSH
• receptory GABA-B genetika   metabolismus   MeSH
• receptory GABA genetika   metabolismus   MeSH
• serin genetika   metabolismus   MeSH
• substituce aminokyselin MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alanin MeSH
• draslík MeSH
• pfetin protein, mouse MeSH Prohlížeč
• proteinkinasy závislé na cyklickém AMP MeSH
• proteiny vázající GTP MeSH
• receptory GABA-B MeSH
• receptory GABA MeSH
• serin MeSH

G-protein-coupled receptors (GPCRs) have been shown to form dimers, but the relevance of this phenomenon in G-protein activation is not known. Among the large GPCR family, metabotropic glutamate (mGlu) receptors are constitutive dimers. Here we examined whether both heptahelical domains (HDs) are turned on upon full receptor activation. To that aim, we measured G-protein coupling efficacy of dimeric mGlu receptors in which one subunit bears specific mutations. We show that a mutation in the third intracellular loop (i3 loop) known to prevent G-protein activation in a single subunit decreases coupling efficacy. However, when a single HD is blocked in its inactive state using an inverse agonist, 2-methyl-6-(phenylethynyl)pyridine (MPEP), no decrease in receptor activity is observed. Interestingly, in a receptor dimer in which the subunit that binds MPEP is mutated in its i3 loop, MPEP enhances agonist-induced activity, reflecting a 'better' activation of the adjacent HD. These data are consistent with a model in which a single HD is turned on upon activation of such homodimeric receptors and raise important issues in deciphering the functional role of GPCR dimer formation for G-protein activation.

• MeSH
• antagonisté excitačních aminokyselin farmakologie   MeSH
• biologické modely MeSH
• buněčné linie MeSH
• dimerizace MeSH
• kinetika MeSH
• lidé MeSH
• mutageneze cílená MeSH
• podjednotky proteinů MeSH
• pyridiny farmakologie   MeSH
• receptory metabotropního glutamátu antagonisté a inhibitory   chemie   genetika   metabolismus   MeSH
• rekombinantní fúzní proteiny antagonisté a inhibitory   chemie   genetika   metabolismus   MeSH
• rekombinantní proteiny antagonisté a inhibitory   chemie   genetika   metabolismus   MeSH
• techniky in vitro MeSH
• terciární struktura proteinů MeSH
• transfekce MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 6-methyl-2-(phenylethynyl)pyridine MeSH Prohlížeč
• antagonisté excitačních aminokyselin MeSH
• metabotropic glutamate receptor type 1 MeSH Prohlížeč
• podjednotky proteinů MeSH
• pyridiny MeSH
• receptory metabotropního glutamátu MeSH
• rekombinantní fúzní proteiny MeSH
• rekombinantní proteiny MeSH