Recept­ors and cel­lu­lar communication

By

Recept­ors and cel­lu­lar communication

G‑Pro­tein-coupled recept­ors – their activ­a­tion, sig­nals and cel­lu­lar localization
Adap­ted from Lohse&Calebiro, Nature 495, 457–458

Over­view

Our group focuses on the mech­an­isms of activ­a­tion and sig­nal­ing of G‑protein coupled recept­ors (GPCR), and sub­sequent down­stream events which lead to alter­a­tions in cell function.

G pro­tein-coupled recept­ors (GPCRs) are a large fam­ily of approx­im­ately 800 mem­bers, which include recept­ors for light, taste and smell, but also many recept­ors for trans­mit­ters and hor­mones. They are also the most import­ant group of tar­gets for drugs, for example beta block­ers, anti­histam­ines and opiates.

All GPCRs share com­mon struc­tural fea­tures, pre­sum­ably also com­mon activ­a­tion mech­an­isms, and they all sig­nal via het­ero­tri­meric G pro­teins (Gαβγ) and – at later points upon activ­a­tion, via β‑arrestins. We use a vari­ety of bio­chem­ical and phar­ma­co­lo­gical meth­ods as well as new micro­scopic tech­niques to study the mech­an­isms of their func­tion and their poten­tial to serve as tar­gets for innov­at­ive drug therapies.

We work closely together with the Receptor Sig­nalling Lab at the Max Del­brück Cen­ter for Molecu­lar Medi­cine in the Helm­holtz Asso­ci­ation (MDC), where large parts of our group are act­ive. Some top­ics and meth­ods are prefer­ably estab­lished there, oth­ers at ISAR Bioscience in Planegg.

Top­ics

Where, when and how fast do recept­ors get activated?

GPCR activ­a­tion is triggered by bind­ing of a lig­and (e.g. adren­aline, glutam­ate) to a spe­cific site of a receptor. This is fol­lowed by a con­form­a­tional change in the receptor, which enables it to bind to and activ­ate G‑proteins. We have developed meth­ods to mon­itor these sig­nalling events via fluor­es­cence micro­scopy, and we aim to study where and when in a cell these sig­nals occur. We use tech­niques to trig­ger receptor activ­a­tion by light to mon­itor sig­nal­ing with sub-mil­li­second res­ol­u­tion. We are also work­ing on tech­niques to adapt such meas­ure­ments to high through­put screen­ing in order to search for com­pounds with unusual sig­nalling prop­er­ties, which might make them new classes of drugs with only a sub­set of effects com­pared to con­ven­tional drugs.

Does cAMP come in nanodomains?

Cyc­lic aden­osine mono­phos­phate (cAMP) is a second mes­sen­ger down­stream of GPCR activ­a­tion, which plays an import­ant role in many physiolo­gical pro­cesses, ran­ging from reg­u­la­tion of heart­beat and force to syn­aptic plas­ti­city. Although cAMP appears to be a freely dif­fus­ible sub­stance, recent evid­ence sug­gests that it may act in cells in a highly loc­al­ized man­ner. We are attempt­ing to visu­al­ize such local effects and are search­ing for mech­an­isms that might lead to the pos­tu­lated nano-com­part­ments. To do so, we use mod­els ran­ging from the design and expres­sion of spe­cific cAMP sensor pro­teins to stud­ies in trans­genic fly and mouse mod­els, where we attempt to image local cAMP signals.

How and why recept­ors make oligomers?

GPCRs can form supra­molecu­lar com­plexes (i.e. di-/oli­gomers) on the cell sur­face. How­ever, their size, nature and dynam­ics as well as their physiolo­gical and phar­ma­co­lo­gical implic­a­tions are still largely unknown. We focus on invest­ig­at­ing the dimer­iz­a­tion of recept­ors (espe­cially adren­er­gic, opi­ate and chemokine) on the sur­face of intact cells as well as eval­u­at­ing the role of dimer­iz­a­tion on receptor activ­a­tion and down­stream sig­nalling. We do so by a vari­ety of bio­chem­ical and micro­scopic tech­niques, not­ably bright­ness ana­lysis and single particle microscopy.

Meth­ods

Epi­fluor­es­cent, con­focal, super-resolution

To track recept­ors or effector pro­teins we use fluor­es­cent labels of dif­fer­ent nature: fluor­es­cent pro­teins, anti­bod­ies, non-organic chem­ic­als, etc. A broad range of micro­scope designs is avail­able in our lab, includ­ing con­focal micro­scope, total internal reflec­tion fluor­es­cence micro­scope, cus­tom-built micro­scope for pho­to­lysis. Micro­scopes are equipped with the new­est gen­er­a­tion of pho­to­de­tect­ors and cam­eras, provid­ing high tem­poral and spa­tial res­ol­u­tion of measurements.

Elec­tronic energy trans­fer through non­ra­di­at­ive dipole–dipole coupling

Major bio­phys­ical tools used in our lab are För­ster Res­on­ance Energy Trans­fer (FRET) and Bio­lu­min­es­cence Res­on­ance Energy Trans­fer (BRET). They allow to meas­ure dis­tances between domains and motifs within or between pro­teins, provid­ing inform­a­tion about receptor con­form­a­tions, G‑protein activ­a­tion, second mes­sen­gers con­cen­tra­tion, etc.

Lumin­es­cence-based drug screen­ing in microtiter plates

Using BRET-based assays adap­ted to microtiter plates we can meas­ure large num­bers of com­pounds and determ­ine their effects on recept­ors and sig­nal cascades.

Bio­chem­ical assays to study pro­tein and nuc­leic acid functions

We have developed numer­ous bio­chem­ical and func­tional assays to char­ac­ter­ize pro­teins and nuc­leic acids involved in sig­nal­ing processes.

Recent Key Publications

Pub­lika­tionen AG Lohse in PubMed