Background and purpose: The CB(1) cannabinoid receptor is regulated by its association with membrane microdomains like the lipid rafts. Here we investigated the role of CB(1) palmitoylation by analyzing the functional consequences of site-specific mutation of cysteine 415, the likely palmitoylated residue at the end of helix 8, in terms of membrane association, raft targeting and signalling of the receptor. Experimental approach: The palmitoylation state of the CB(1) receptor was assessed in rat forebrain by depalmitoylation/repalmitoylation experiments. Cysteine 415 was replaced with alanine by site directed mutagenesis. Green fluorescence protein chimeras of both wild-type and mutant receptors were transiently expressed and functionally characterised in SH-SY5Y cells and HEK-293 cells by means of confocal microscopy, cytofluorimetry and competitive binding assays. Confocal FRAP was used to assess receptor membrane dynamics, whereas [(35) S]GTPγS, cAMP and co-immunoprecipitation assays were employed to assess signalling activity. Key results: Endogenous CB(1) receptors were palmitoylated in rat brain. Mutation of cysteine 415 in transfected cells prevented the palmitoylation of the receptor and significantly reduced it recruitment at both plasma membrane and lipid rafts; it also increased protein diffusional mobility. The same mutation markedly reduced the functional coupling of CB(1) receptor with G proteins and adenylyl cyclase, whereas depalmitoylation abrogated receptor association with a specific subset of G proteins. Conclusions and implications: We found that CB(1) receptor is post-translationally modified by palmitoylation and that mutation of cysteine 415 gives rise to a form of receptor that is functionally impaired in terms of membrane targeting and signalling.[...]
Effects of palmitoylation of Cys 415 in helix 8 of the CB1 cannabinoid receptor on membrane localization and signalling
CATANZARO, Giuseppina;
2012-01-01
Abstract
Background and purpose: The CB(1) cannabinoid receptor is regulated by its association with membrane microdomains like the lipid rafts. Here we investigated the role of CB(1) palmitoylation by analyzing the functional consequences of site-specific mutation of cysteine 415, the likely palmitoylated residue at the end of helix 8, in terms of membrane association, raft targeting and signalling of the receptor. Experimental approach: The palmitoylation state of the CB(1) receptor was assessed in rat forebrain by depalmitoylation/repalmitoylation experiments. Cysteine 415 was replaced with alanine by site directed mutagenesis. Green fluorescence protein chimeras of both wild-type and mutant receptors were transiently expressed and functionally characterised in SH-SY5Y cells and HEK-293 cells by means of confocal microscopy, cytofluorimetry and competitive binding assays. Confocal FRAP was used to assess receptor membrane dynamics, whereas [(35) S]GTPγS, cAMP and co-immunoprecipitation assays were employed to assess signalling activity. Key results: Endogenous CB(1) receptors were palmitoylated in rat brain. Mutation of cysteine 415 in transfected cells prevented the palmitoylation of the receptor and significantly reduced it recruitment at both plasma membrane and lipid rafts; it also increased protein diffusional mobility. The same mutation markedly reduced the functional coupling of CB(1) receptor with G proteins and adenylyl cyclase, whereas depalmitoylation abrogated receptor association with a specific subset of G proteins. Conclusions and implications: We found that CB(1) receptor is post-translationally modified by palmitoylation and that mutation of cysteine 415 gives rise to a form of receptor that is functionally impaired in terms of membrane targeting and signalling.[...]I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
