Multimerization Studies of Membrane Receptors via μ-Patterned Surfaces

Abstract

Studying dimerization or multimerization processes of plasma-membrane localized receptor proteins is important for a better understanding of various cellular processes. While the physiological role of receptor multimerization remains a matter of debate for many proteins, it has been proved that such events are crucial for the function of different classes of membrane receptors (e.g. EGF or Insulin receptor). Several biochemical tools including cross-linking or co-IP assays are used to unravel protein multimerization. However, these techniques suffer from being indirect and hardly quantitative and give high number of false-positives and/or negatives. Indeed, there are biophysical assays to address this question, but the evaluation of the recorded data remains complicated and laborious. Here we describe how a combination of Total Internal Reflection Fluorescence (TIRF) microscopy and micro-structured surfaces (“micropatterning assay”) can be used to easily prove multimerization of membrane receptors. This technique was developed to detect protein-protein interactions (Schwarzenbacher et al., 2008; Weghuber et al., 2010) and offers the possibility to measure and quantify also weak or short-lived interactions in vivo. In a proof of concept experiment we studied the multimerization of YFP and/or CFP labeled s1 and s2 adrenergic receptors. The question of homo- and especially hetero-association of these G-protein coupled receptors (GPCR) is far from being completely understood. Receptor maturation, G-protein coupling, downstream signaling and regulatory processes such as internalization might be influenced by dimerized adrenergic receptors. We unequivocally show that s1 as well as s2 receptors form homo- and hetero multimers in living CHO-K1 cells. Our findings contribute to the field of adrenergic receptors. In general, our system might be of great interest for a fast and straightforward analysis of membrane-protein multimerization.