Identification and characterization of CRIP1b: a novel CB₁ cannabinoid receptor interaction protein

Abstract

G protein-coupled receptors. (GPCRs) transduce extracellular stimuli to intracellular signals. through their interaction with heterotrimeric G proteins. Signaling diversity and specificity is imparted primarily through variations of G protein subunits. Protein-protein interactions between intracellular accessory proteins and GPCRs also modify signaling by altering receptor activity or signaling pathways. The ability of intracellular proteins to interact with the CB 1 cannabinoid receptor was investigated to determine whether particular signaling properties of CB 1 resulted from interaction with specific CB 1 interacting proteins. A novel protein named CRIP 1 b was discovered· to interact with the C-terminal tail of CB 1 ~- The interaction between CRIP 1 b and CB 1 was characterized using the yeast two-hybrid assay. Functional consequences of the CRIPlbCB 1 interaction were investigated by examining protein localization by confocal microscopy and measuring CB1 mediated N-type Ca2+ channel activity in the presence of CRIP 1 b by whole-cell patch clamp recordings. The ye_ast two-hybrid assay indicated that the last nine amino acids of the. CB 1 C-terminal tail were required for interaction with CRIP 1 b. Heterologous expression of CRIP 1 b and CB 1 in HEK 293 cells did not reveal evidence of co localization, nor was CB 1 able to significantly traffic CRIP 1 b to the plasma membrane. However, CRIP 1 b and CB1 were found to colocalize in superior cervical ganglion (SCG) neurons. Whole-cell voltage-clamp recordings ofN-type Ca2+ channels in SCG neurons indicated that CRIPlb had no.effect on agonist- or inverse agonist-induced modulation of Ca2

current by CB1. Furthefrr1:ore, the level of CB 1 constitutive activity was· not significantly altered by CRIP 1 b. The high affinity of CB 1 for G proteins, as demonstrated by the ability of CB 1 to sequester G proteins from other Gu0 coupled receptors, was unaffected by expression ofCRIPlb. These results provide evidence that CRIP 1 b is a novel CB 1 accessory protein that interacts with the C-terminal tail of CB1. While CRIPlb and CB1 can apparently interact in a neuronal expression system, the ability of CRIP 1 b to modify CB 1 signaling was not detected in any of the pathways investigated. Thus, the distinctive signaling properties of CB1, such as constitutive activity and G protein seque.stration do not originate from nor are modified by CRIPlb.