The CB1 cannabinoid receptor : receptor states, activity and G protein sequestration

Abstract

The human CB I cannabinoid receptor is a member of the G prot~in coupled ' . •,' . ' receptor family. Th~ CB I cannabinoid ~eceptor couples to pertussis toxin sensitive Gi/o proteins and ·inhibits ri~uronal voltage-gated Ca2+· channels. The. hCB I r~ceptor has two . . - - ,·' i unusual properties: I) it is constitutively active in the absence ofago~~st and 2) it can prevent other G protein coupled receptors from signaling by sequestering: a common pool of Gi/o proteins. The mechanism of consJitutive activity and G protein ~equestration by the hCB I receptor is unknown. In this ·.stud~, two Cijrboxyl terminal truncation mutants (hCBI-417 and ~CBI-400) were used to test the hypothesis that the pnj,ximal carboxyl. ' ' ' terminal couples to G proteins while the distal ·carboxyl terminal modulates G protein j sequestration and constitutive activity. Additionally, mutation of a sing~e amino acid in the second transmembrane domain (rCB 1-D 164N) was used to test the: hypothesis that this amino acid plays a critical role in tlie structural basis of G protein coupling and constitutive activity: Receptor cDNA constructs were injected_ into the nucleus of s~perior cervical ganglion neurons. After an overnight incubation to allow for receptor expression, neurons were voltage clamped and Ca2+ current were recorded. Inhibition of the; Ca2+ current by the cannabinoid agonist WIN 55,212-2 was used as an index of C~l cannabinoid receptor G. protein coupling and activation. Ca2+ channels are inhibited by Gfly subunits_ -released from· activated Gi/o proteins. In contrast to the wild type C.B 1 cannabinoid receptor, the mutant receptor in which the entire carboxyl terminal (amino acids 401-472) was deleted (hCB 1-400) failed to inhibit the Ca2+ current. Deletion of only the distal carboxyl terminal ( amino acids 418-4 72; hCB 1-417) restored Ca2+ current inhibition. These results demonstrate the critical role of the proximal domain (amino acids 401-417) ' I of the carboxyl terminal of the hCB 1 receptor in coupling to G proteins. ; Truncation of the distal carboxyl terminal domain,· however, Ghanged the magnitude of Ca2+ current inhibition. The hCB 1-417 receptor produced significantly less inhibition of the Ca2+ current in the presence of WIN 55,212-2 c9~pared to the wild typ~- •. receptor (22.6±3.0% vs 43.7±6.5%, respectively). Thus, the distal c~rboxyl terminal domain is important in modulating the magnitude of Ca2+ current inhib~tion. In addition to the change in the magnitude of Ca2+ current inhibition, deletion of th¢ distal carboxyl terminal significantly slowed the kinetics of Ca2+ current inhibition byi WIN 55,212-2 (time to peak of effect: 146.0±8.7 second). The distal carboxyl terminal tail of the CBI canriabinoid receptor also played a· role in constitutive activity ~nd G protein : sequestration. The hCBI-417 receptor displayed. enhanced constitut~ve activity. In neurons injected with 50 ng/μl hCBI-417 cDNA the inverse agonist SR141716A increased the Ca2+ .current 101.1±21.3%. The inverse agonist acts ~to reverse the·. constitutive activity of the receptor. The effect of SR141716A on the hCBl-417 receptor was significantly_ greater than the 42.9±7.6% Ca2+ current increase in neu;rons expressing the wild type hCBI receptor .. · G protein sequestration was also enhanced in neurons expressing the truncated hCBI-417 receptor. Wild type h~Bl cannabinoid receptors when expres:sed by injecting · 100 ng/μl cDNA completely abolish signa~ing by other G protein coupled receptors including a2-adrenergic receptors. Normally activation of a 2-adrenergic r~ceptors inhibits the Ca2 + current 44.5±5.7%. In the presence of hCBI receptors activ~tion of the a 2.: adrenergic receptors by UK.14304 inhibited the Ca2+ current only 1.5±4.2%. Signaling by a2-adrenergic receptors can be partially restored by injecting a lower concentration of hCBI cDNA. In neurons injected with 50 ng/μl hCBl cDNA the a2-adrenergic agonist UK.14304 inhibited the Ca2+ current 20.0±3.7%. In neurons injected with: 50 ng/μl hCBI- 417 cDNA UK.14304 inhibited the Ca2+ current 7.0±1.2%. Thus, sign~lilJ,g by the a 2- _adrenergic receptor was abolished by the catboxyl terminal truncated hC~l-417 receptor. These results indica,te that deletion ~f the distal carboxyl terminal enhances the ability of the receptor to sequester· G proteins. ! The aspartic acid residue in the s~cond transmembr~ne_ dom~in of G protein ,.· ' . ; coupled receptors is highly conserved. -Mutation of this aspartic acid (r~Bl-D164N) had profound effects on the constitutive activity of the CBI receptor as well :as.on the abilityof the receptor to sequester G proteins. Both the constitutive activity mid the ability to sequester G proteins were abolished by the rCBI-D164N· ieceptor. The inverse agonist increased the Ca2 + current only 11.6±6.9% in neurons expressing the mutant rCBl- : D164N receptors. The mutant rCB1-D164N receptors failed to block signaling by the a 2- adrengic receptor. UK.14304 inhibited the Ca2+ current 35.8±6.8% in neurons expressing the rCB l-Dl64N receptors and was not different from control neurons. Additionally, the D 164N mutation in the second transmembrane domain decreased the tim.e to peak of the WIN 55,212-2 inhibition of the Ca2+ current to 24±4 seconds. These results demonstrate that 1) the proximal carboxyl terminal domain of the hCB 1 receptor is critical for G protein coupling, 2) the distal carboxyl tbrminal domain regulates constitutive activity, G protein coupling kinetics and G protein sequestration and _3) the aspartic acid in the second tra~smembrane domain plays a critical role in G . ' 1 . protein sequestration, G protein coupling_ kinetics and constitutive activ~ty of the hCB 1 receptor. Taken together, the mutant receptors shift the CB 1 recept~r into different receptor states. The D164N-CB1 receptor exists primarily in an inactive'.state uncoupled from G proteins, the wild type CB 1 receptor e~sts in both active and inactive G protein coupled states and the carboxyl terminal truncated CB 1-417 receptor exists primarily in an active G protein coupled state.