Structural determinants in the second intracellular loop of the human cannabinoid CB1 receptor mediate selective coupling to G(s) and G(i)

Br J Pharmacol. 2010 Dec;161(8):1817-34. doi: 10.1111/j.1476-5381.2010.01006.x.


Background and purpose: The cannabinoid CB(1) receptor is primarily thought to be functionally coupled to the G(i) form of G proteins, through which it negatively regulates cAMP accumulation. Here, we investigated the dual coupling properties of CB(1) receptors and characterized the structural determinants that mediate selective coupling to G(s) and G(i).

Experimental approach: A cAMP-response element reporter gene system was employed to quantitatively analyze cAMP change. CB(1)/CB(2) receptor chimeras and site-directed mutagenesis combined with functional assays and computer modelling were used to determine the structural determinants mediating selective coupling to G(s) and G(i).

Key results: CB(1) receptors could couple to both G(s)-mediated cAMP accumulation and G(i)-induced activation of ERK1/2 and Ca(2+) mobilization, whereas CB(2) receptors selectively coupled to G(i) and inhibited cAMP production. Using CB(1)/CB(2) chimeric receptors, the second intracellular loop (ICL2) of the CB(1) receptor was identified as primarily responsible for mediating G(s) and G(i) coupling specificity. Furthermore, mutation of Leu-222 in ICL2 to either Ala or Pro switched G protein coupling from G(s) to G(i), while to Ile or Val led to balanced coupling of the mutant receptor with G(s) and G(i) .

Conclusions and implications: The ICL2 of CB(1) receptors and in particular Leu-222, which resides within a highly conserved DRY(X)(5) PL motif, played a critical role in G(s) and G(i) protein coupling and specificity. Our studies provide new insight into the mechanisms governing the coupling of CB(1) receptors to G proteins and cannabinoid-induced tolerance.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3T3 Cells
  • Adenylyl Cyclases / drug effects
  • Animals
  • CHO Cells
  • COS Cells
  • Calcium / metabolism
  • Cell Line, Transformed
  • Chlorocebus aethiops
  • Cricetinae
  • Cricetulus
  • Cyclic AMP / metabolism
  • Extracellular Signal-Regulated MAP Kinases / metabolism
  • GTP-Binding Protein alpha Subunits, Gi-Go / metabolism*
  • GTP-Binding Protein alpha Subunits, Gs / metabolism*
  • HEK293 Cells
  • Humans
  • Mice
  • Mutagenesis, Site-Directed / methods
  • Protein Binding / genetics*
  • Protein Interaction Domains and Motifs*
  • Receptor, Cannabinoid, CB1 / genetics
  • Receptor, Cannabinoid, CB1 / metabolism*
  • Receptor, Cannabinoid, CB2 / genetics
  • Receptor, Cannabinoid, CB2 / metabolism


  • Receptor, Cannabinoid, CB1
  • Receptor, Cannabinoid, CB2
  • Cyclic AMP
  • Extracellular Signal-Regulated MAP Kinases
  • GTP-Binding Protein alpha Subunits, Gi-Go
  • GTP-Binding Protein alpha Subunits, Gs
  • Adenylyl Cyclases
  • Calcium