Cannabinoid receptor interacting protein 1a (CRIP1a) is a CB receptor (CBR) distal C-terminal-associated protein that alters CBR interactions with G-proteins. We tested the hypothesis that CRIP1a is capable of also altering CBR interactions with β-arrestin proteins that interact with the CBR at the C-terminus. Coimmunoprecipitation studies indicated that CBR associates in complexes with either CRIP1a or β-arrestin, but CRIP1a and β-arrestin fail to coimmunoprecipitate with each other. This suggests a competition for CRIP1a and β-arrestin binding to the CBR, which we hypothesized could attenuate the action of β-arrestin to mediate CBR internalization. We determined that agonist-mediated reduction of the density of cell surface endogenously expressed CBRs was clathrin and dynamin dependent and could be modeled as agonist-induced aggregation of transiently expressed GFP-CBR. CRIP1a overexpression attenuated CP55940-mediated GFP-CBR as well as endogenous β-arrestin redistribution to punctae, and conversely, CRIP1a knockdown augmented β-arrestin redistribution to punctae. Peptides mimicking the CBR C-terminus could bind to both CRIP1a in cell extracts as well as purified recombinant CRIP1a. Affinity pull-down studies revealed that phosphorylation at threonine-468 of a CBR distal C-terminus 14-mer peptide reduced CBR-CRIP1a association. Coimmunoprecipitation of CBR protein complexes demonstrated that central or distal C-terminal peptides competed for the CBR association with CRIP1a, but that a phosphorylated central C-terminal peptide competed for association with β-arrestin 1, and phosphorylated central or distal C-terminal peptides competed for association with β-arrestin 2. Thus, CRIP1a can compete with β-arrestins for interaction with C-terminal CBR domains that could affect agonist-driven, β-arrestin-mediated internalization of the CBR.

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A previous study from this laboratory demonstrated that treatment of pregnant mice with 3-methylcholanthrene (MC) caused lung tumors in the offspring at 1 year after birth, the incidence of which correlated with fetal inducibility of Cyp1a1. Analysis by PCR amplification and allele-specific hybridization (ASO) of paraffin-embedded tumors generated from that study revealed the presence of point mutations in exon 1 of the Ki-ras gene. This work has now been expanded by PCR amplification and ASO analysis of 31 additional lesions. Point mutations were found in 37 of the 47 (79%) lesions analyzed in this and the previous study, the majority of which were G-->T transversions in the first or second base of codon 12. The mutational spectrum appeared to be dependent on the relative stage of differentiation of the lesion, as both the incidence of mutation and type of mutation produced correlated with malignant progression. Mutations occurred in 60% of the hyperplasias, 80% of the adenomas and 100% of the adenocarcinomas. In the lesions with mutations, GLY12-->CYS12 transversions occurred in 100% of the hyperplasias, 42% of the adenomas and 14% of the adenocarcinomas. The GLY12-->VAL12 transversions occurred in none of the hyperplasias, 42% of the adenomas and 57% of the adenocarcinomas. The remaining mutations, which consisted of ASP12 transitions and ARG13 transversions, occurred only in adenomas (17%) and adenocarcinomas (29%). Between this study and our previous analyses, the identity of the mutations obtained by ASO were confirmed by sequence analysis of eight of the 37 lesions that harbored mutations at the Ki-ras gene locus. There were no differences in the type or incidence of mutations relative to the metabolic phenotype or sex of the mice. These data suggest that mutational activation of the Ki-ras gene locus is an early event in transplacental lung tumorigenesis, and that the type of mutations produced by exposure to chemical carcinogens can influence the carcinogenic potential of the tumor. This may have prognostic significance in determining the malignant progression of the neoplasm.