Mechanism And Role Of AR Plasma Membrane Translocation

The androgen receptor(AR)is a member of the Type I nuclear receptor subfamily.AR plays an important role in the development and maintenance of the male sexual phenotype.Similar to other steroid hormone receptors,AR is located in the cytoplasm in the absence of androgens,forming a complex with the chaperones and co-chaperones such as heat shock proteins and immunophilins.Upon androgen binding,AR undergoes conformational changes and translocates to the nucleus,activates target genes expression,and induces cell proliferation,differentiation,and survival.This mode of action is the classical AR signaling pathway,also known as the genomic action.In addition to the classical signaling pathway,many observations suggest androgen,as well as some other steroid hormones,can affect cellular processes in a non-genomic fashion.AR activates the PI3K-AKT signaling pathway,ERK singaling pathway and Src signaling pathway via the non-genomic function of AR.As more and more studies focus on the non-genomic AR signaling pathway,researches found androgen induces AR membrane transport besides the nuclear transport.And AR membrane transport was involved in the activation of AKT.AR interacted with AR on the cell plasma membrane.Interrupting AR plasma membrane transport inhibited the AKT phosphorylation,indicating AR plasma membrane transport plays an important role in AR function.Studies found overexpression caveolin-1 enhanced AR membrane localization,suggesting caveolin-1 was involved in AR membrane transport.However,AR and caveolin-1 were not co-expressed in majority of prostate cancer cell lines,indicating caveolin-1 might facilitate AR membrane transport but not mediate AR membrane transport.It's still unknown how AR translocates to plasma membrane.Several studies on AR nuclear transport clarified that AR nuclear transport was microtubule-depend and mediated by the motor protein dynein,and further transport through the nuclear pore complex by importin ?/?family.So is AR membrane transport is based on microtubule function? If AR membrane transport is microtubule dependent,which motor protein mediates this transport? And besides the activation of AKT,what else can membrane-located AR regulate?To answer these questions,several assay were applied.We first demonstrated androgen induced AR rapidly plasma membrane transport by using immunofluorescence and plasma membrane fractionation assay,and also found the androgen induction of AR membrane transport abolished by androgen antagonist,suggesting androgen induced AR membrane transport.We quantitatively analyzed the localization of AR plasma membrane,by using sucrose density gradient centrifugation.It was found that about 8% of AR transported to the plasma membrane under the conditions stimulation 20 min of androgen,demonstrating that androgen induced a rapid AR plasma membrane transport.It was found by using microtubule-targeted drug docetaxel,which inhibited microtubule depolymerization or nocodazole,which inhibited the stabilization of microtubules inhibited AR plasma membrane localization,and interrupting actin function by cytochalasin D had no effect on the AR plasma membrane translocation,demonstrating that the plasma membrane translocation of AR was also dependent on microtubules.Further I found that KIF5 B acted as a kinesin to mediate microtubuledependent AR trafficking.Inhibition of KIF5 B by a dominant-negative mutant or knockdown of KIF5 B by siRNA suppressed the plasma membrane translocation of AR.Simultaneous interference with the function of KIF5 B also inhibited the activation of downstream AKT,demonstrating that KIF5 B participated in the AR plasma membrane transport.If KIF5 B was involved in AR transport as a kinesin motor protein,there must be direct interaction between AR and KIF5 B.By applying immunoprecipitation experiments,it was found that there is an interaction between AR and KIF5 B,and androgen enhanced interaction between AR and KIF5 B.By constructing deletions of different AR functional domains,I further indentified that AR interacted with KIF5 B through its N-terminal domain.Based on the above experiments,it is fully demonstrated that the motor protein KIF5 B mediated the AR plasma membrane transport.The palmitoylation modification is important for the protein plasma membrane localization.The steroidal nuclear receptor family members had been shown to be palmitoylated,and palmitoylation of AR occurs at the 807 site cysteine.By using 2-bromopalmitate to inhibit of AR-palmitoylation or mutation of AR palmitoylation site(AR-C807A)effectively inhibited AR plasma membrane localization,but had no effect on regulating the interaction between AR and KIF5 B,suggesting that palmitoylation may work as AR plasma membrane localization signal but not involved in the transport process of AR.I further applied the functional studies of AR,and found that interference with AR plasma membrane transport inhibited induction of AR target genes PSA and TMRPSS2 mRNA by androgen.The data showed that AR-C807A(a plasma membrane location deficient AR)was not only losing the ability to locate in the plasma membrane,but it also showed a decreasing in nuclear transport by using immunofluorescence assay.Further by nuclear cytoplasmic fractionation assay,it confirmed that AR-C807 A had low nuclear transport efficiency.The above experiments showed that 8% of AR that undergo to the plasma membrane regulate the nuclear transport of rest AR,regulate the transcriptional activities,and regulate the genome functions.Wild-type AR rapidly induces the phosphorylation of HSP27 under the action of androgen.Phosphorylated-HSP27 further associates with AR and facilitate nuclear transport of AR.Here,I found that AR-C807 A didn't induce the phosphorylation of HSP27 under the action of androgen,suggesting that the plasma membrane transport AR maybe regulate AR nuclear transport via HSP27.To further confirm the role of HSP27,I co-expressed HSP27-mimic(phosphorylated HSP27)and AR-C807 A in DU145 cells and found that HSP27-mimic rescued the expression of AR target gene TMRPSS2 and rescued partial of the cell proliferation.It is fully proved that the plasma membrane transport AR regulates the genomic function of AR by activating HSP27,regulates cell proliferation and target gene expression.To summarize the above experiments,I study the mechanism and role of the plasma membrane transport AR in this thesis.It is found that AR translocates to the plasma membrane in a microtubule-dependent manner.The motor protein KIF5 B mediates the transport by binding to the N-terminal domain of AR.The plasma membrane transport AR regulates the activation of AKT,phosphorylates HSP27,and thereby regulating the nuclear transport and transcriptional activity of AR.The above studies provide important experimental data for improving the pathway of androgen receptor signaling.At the same time,AR plasma membrane translocation occurs at very low level of androgen(castrated level of androgen),suggesting that AR plasma membrane translocation maybe play an important role in processing of castration resistant prostate cancer.The study also provides new molecular mechanisms of castration resistant prostate cancer.