Mapping the structural requirements in the CB1 cannabinoid receptor transmembrane helix II for signal transduction. 2008

Ankur Kapur, and Patrick Samaniego, and Ganesh A Thakur, and Alexandros Makriyannis, and Mary E Abood
California Pacific Medical Center Research Institute, San Francisco, California, USA.

Amino acid residues in the transmembrane domains of the CB(1) receptor are important for ligand recognition and signal transduction. We used site-directed mutagenesis to identify the role of two novel and adjacent residues in the transmembrane helix II domain, Ile2.62 and Asp2.63. We investigated the role of the conserved, negatively charged aspartate at position 2.63 in cannabinoid receptor (CB(1)) function by substituting it with asparagine (D2.63N) and glutamate (D2.63E). In addition, the effect of the mutant I2.62T alone and in combination with D2.63N (double mutant) on the affinity and potency of structurally diverse ligands was investigated. Recombinant human CB(1) receptors, stably expressed in human embryonic kidney 293 cells, were assayed for ligand affinity and agonist-stimulated guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding. The charge-conserved mutant D2.63E behaved similar to wild type. The charge-neutralization mutation D2.63N attenuated the potency of (-)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)phenyl]-4-[3-hydroxylpropyl] cyclohexan-1-ol (CP,55940), (R)-(-)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl)methanone (WIN55212-2), (-)-11beta-hydroxy-3-(1',1'-dimethylheptyl) hexahydrocannabinol (AM4056), and (-)-11-hydroxyldimethylheptyl-Delta(8)-tetrahydrocannabinol (HU210) for the stimulation of GTPgammaS binding, without affecting their binding affinities. Likewise, the I2.62T mutant selectively altered agonist potency without altering agonist affinity. It was surprising to note that the double mutant (I2.62T-D2.63N) displayed a drastic and synergistic increase (by approximately 50-fold) in the EC(50) for agonist-mediated activation. The profound loss of function in the I2.62T-D2.63N double mutant suggests that, although these residues are not obligatory for agonist recognition, they play a synergistic and crucial role in modulating signal transduction.

UI MeSH Term Description Entries
D011485 Protein Binding The process in which substances, either endogenous or exogenous, bind to proteins, peptides, enzymes, protein precursors, or allied compounds. Specific protein-binding measures are often used as assays in diagnostic assessments. Plasma Protein Binding Capacity,Binding, Protein
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D001216 Asparagine A non-essential amino acid that is involved in the metabolic control of cell functions in nerve and brain tissue. It is biosynthesized from ASPARTIC ACID and AMMONIA by asparagine synthetase. (From Concise Encyclopedia Biochemistry and Molecular Biology, 3rd ed) L-Asparagine
D001665 Binding Sites The parts of a macromolecule that directly participate in its specific combination with another molecule. Combining Site,Binding Site,Combining Sites,Site, Binding,Site, Combining,Sites, Binding,Sites, Combining
D015398 Signal Transduction The intracellular transfer of information (biological activation/inhibition) through a signal pathway. In each signal transduction system, an activation/inhibition signal from a biologically active molecule (hormone, neurotransmitter) is mediated via the coupling of a receptor/enzyme to a second messenger system or to an ion channel. Signal transduction plays an important role in activating cellular functions, cell differentiation, and cell proliferation. Examples of signal transduction systems are the GAMMA-AMINOBUTYRIC ACID-postsynaptic receptor-calcium ion channel system, the receptor-mediated T-cell activation pathway, and the receptor-mediated activation of phospholipases. Those coupled to membrane depolarization or intracellular release of calcium include the receptor-mediated activation of cytotoxic functions in granulocytes and the synaptic potentiation of protein kinase activation. Some signal transduction pathways may be part of larger signal transduction pathways; for example, protein kinase activation is part of the platelet activation signal pathway. Cell Signaling,Receptor-Mediated Signal Transduction,Signal Pathways,Receptor Mediated Signal Transduction,Signal Transduction Pathways,Signal Transduction Systems,Pathway, Signal,Pathway, Signal Transduction,Pathways, Signal,Pathways, Signal Transduction,Receptor-Mediated Signal Transductions,Signal Pathway,Signal Transduction Pathway,Signal Transduction System,Signal Transduction, Receptor-Mediated,Signal Transductions,Signal Transductions, Receptor-Mediated,System, Signal Transduction,Systems, Signal Transduction,Transduction, Signal,Transductions, Signal
D016415 Sequence Alignment The arrangement of two or more amino acid or base sequences from an organism or organisms in such a way as to align areas of the sequences sharing common properties. The degree of relatedness or homology between the sequences is predicted computationally or statistically based on weights assigned to the elements aligned between the sequences. This in turn can serve as a potential indicator of the genetic relatedness between the organisms. Sequence Homology Determination,Determination, Sequence Homology,Alignment, Sequence,Alignments, Sequence,Determinations, Sequence Homology,Sequence Alignments,Sequence Homology Determinations
D017124 Conserved Sequence A sequence of amino acids in a polypeptide or of nucleotides in DNA or RNA that is similar across multiple species. A known set of conserved sequences is represented by a CONSENSUS SEQUENCE. AMINO ACID MOTIFS are often composed of conserved sequences. Conserved Sequences,Sequence, Conserved,Sequences, Conserved
D017433 Protein Structure, Secondary The level of protein structure in which regular hydrogen-bond interactions within contiguous stretches of polypeptide chain give rise to ALPHA-HELICES; BETA-STRANDS (which align to form BETA-SHEETS), or other types of coils. This is the first folding level of protein conformation. Secondary Protein Structure,Protein Structures, Secondary,Secondary Protein Structures,Structure, Secondary Protein,Structures, Secondary Protein
D043884 Receptor, Cannabinoid, CB1 A subclass of cannabinoid receptor found primarily on central and peripheral NEURONS where it may play a role modulating NEUROTRANSMITTER release. Cannabinoid Receptor CB1,CB1 Receptor,Receptor CB1, Cannabinoid,Receptor, CB1
D019943 Amino Acid Substitution The naturally occurring or experimentally induced replacement of one or more AMINO ACIDS in a protein with another. If a functionally equivalent amino acid is substituted, the protein may retain wild-type activity. Substitution may also diminish, enhance, or eliminate protein function. Experimentally induced substitution is often used to study enzyme activities and binding site properties. Amino Acid Substitutions,Substitution, Amino Acid,Substitutions, Amino Acid

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