Chapter

Secretin Receptor Dimerization: A Possible Functionally Important Paradigm for Family B G Protein-Coupled Receptors*

KALEECKAL G. HARIKUMAR, MAOQING DONG, and LAURENCE J. MILLER

Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, AZ

Protein-protein interactions represent a molecular mechanism for signal propagation and for regulation that is utilized extensively in cell biology and physiology. The interaction between single transmembrane tyrosine kinase receptors has long been recognized as a fundamental mechanism for the cross-phosphorylation involved in regulation of the activity of these important growth regulators [ 1] . After a long history of being considered as acting as single units, guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) have recently also been reported to form dimers or higher. order oligomers [2]. However, the extent that this process occurs in normal physiology and its functional significance have been quite controversial [3, 4].

In this report, we will review the methodological approaches that have been utilized to demonstrate GPCR oligomerization, the structural themes for such association, and the functional effects that have been proposed. Family B GPCR dimerization may represent a unique and structurally specific process that can have clear functional implications. Within this chapter, we will carefully review the data relevant to the secretin receptor as a possible paradigm for how this process might affect Family B GPCRs, as well as the larger GPCR superfamily.

*This work was supported by grants from the National Institutes of Health, DK46577 (LJM), the Fiterman Foundation, and the Mayo Clinic.

GPCR Molecular Pharmacology and Drug Targeting: Shifting Paradigms and New Directions,

Edited by Annette Gilchrist

Copyright © 2010 John Wiley & Sons, Inc.

6.1. METHODOLOGICAL APPROACHES TO GPCR OLIGOMERIZATION

GPCR oligomerization has been explored a number of ways, including approaches dependent on structural associations and those involving functional reconstitution. It should be mentioned that procedures have often utilized the high- l evel expression of recombinant proteins, but such a level of expression may induce artificial interactions that might not be present in a physiological environment. Therefore, when possible, use of natural receptor-bearing cells or those expressing receptors at physiologic levels are preferred. The oldest approach to demonstrate physical association is the co-immuno-precipitation (co-IP) of two receptors, utilizing an antibody to one receptor to precipitate the partner receptor [5] . Since there is a paucity of high-quality, highly selective, high-affinity antibodies to natural receptors that are currently available, most of these studies have utilized epitope-tagged forms of receptors coexpressed in heterologous cell lines. This also permits the examination of receptor homodimerization, with the same receptor being prepared as two distinctly tagged forms. Controls to ensure adequate solubilization including the ultracentrifugation of the extracts or their passage through high-resolution filters prior to immunoprecipitation have been helpful, as have controls that involve the mixing of detergent extracts from two cell lines, each expressing only one receptor; however, this technique is still criticized and cannot be utilized as evidence of receptor oligomerization without the existence of complementary data utilizing another distinct technique. This relates to the tendency of these hydrophobic proteins to aggregate and to nonspecifically associate, as well as to the possibility that a detergent micelle might include multiple nonphysically associated membrane proteins.

The most common complementary technique involves resonance energy transfer, with nonradiative transfer of energy between a donor attached to one receptor and an acceptor attached to a second receptor (Fig. 6.1). This can utilize bioluminescence resonance energy transfer (BRET) [6] or fluorescence resonance energy transfer (FRET) [7]. The latter has even been utilized as a quantitative approach to measure distances between donor and acceptor [8] . BRET has a theoretical advantage over FRET, since donor effects on the acceptor signal are less and do not require correction. By choice of donor and acceptor with the appropriate overlap of donor emission and acceptor absorption, the resonance energy transfer signal can be limited to relatively short distances, but those that are adequate to identify complexes of physically associated receptors. Nevertheless, at high levels of engineered recombinant receptor expression, it becomes easy to visualize crowding of the lipid bilayer resulting in the generation of nonspecific signals in such studies. This phenomenon has been termed "nonspecific bystander effect" i 9] . To overcome this potential problem, saturation resonance transfer techniques have been employed in which varied ratios of donor-tagged and acceptor-tagged receptors are coexpressed and evaluated for resonance transfer [9]. In such studies,

--Acceptor

FRET

-Donor

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Wavelength (nm)

Figure 6.1 Study of secretin receptor oligomerization by fluorescence resonance energy transfer techniques. Left panel shows a representative confocal microscopic image of a COS (African green monkey kidney) cell expressing the YFP-tagged secretin receptor construct, demonstrating normal trafficking of the fluorescently tagged receptors to the cell surface. Right panel shows representative fluorescence emission spectra, including spectra of the donor (human secretin receptor-cyan fluorescent protein [HSecR-CFP]) excited at 433 nm, the acceptor (HSecR-YFP) excited at the donor excitation wavelength (433 nm) and excited at the wavelength optimal to elicit acceptor emission (480 nm), and a typical FRET emission profile from a system containing both donor and acceptor that was stimulated at 433 nm. Bar, 25 |im.

04450

--Acceptor

FRET

-Donor

Wavelength (nm)

Figure 6.1 Study of secretin receptor oligomerization by fluorescence resonance energy transfer techniques. Left panel shows a representative confocal microscopic image of a COS (African green monkey kidney) cell expressing the YFP-tagged secretin receptor construct, demonstrating normal trafficking of the fluorescently tagged receptors to the cell surface. Right panel shows representative fluorescence emission spectra, including spectra of the donor (human secretin receptor-cyan fluorescent protein [HSecR-CFP]) excited at 433 nm, the acceptor (HSecR-YFP) excited at the donor excitation wavelength (433 nm) and excited at the wavelength optimal to elicit acceptor emission (480 nm), and a typical FRET emission profile from a system containing both donor and acceptor that was stimulated at 433 nm. Bar, 25 |im.

random collisions between bystanders are expected to increase in a linear manner with an increasing ratio of acceptor/donor, while significant structurally specific molecular associations would be expected to result in hyperbolic curves in which resonance energy transfer would approach a maximum level, as all available donors become complexed with the acceptors. Because of the requirement of fluorescent or luminescent tags that are not naturally present on receptors, these methods are not applicable to natural cellular systems in which there are normal levels of expression of receptors and relevant regulatory proteins. Receptor association might be suggested indirectly in such systems by demonstration of cooperativity of binding or action.

Other techniques have also been applied less commonly to explore receptor oligomerization. These include the stimulation of the internalization of one receptor by the treatment with an agonist of the second receptor [ 10] , the trapping of one receptor in the biosynthetic cascade by the expression of a dominant negative partner receptor [11], rescuing an intracellularly trapped receptor by expression of another partner receptor [12] , the modification of the binding properties of one receptor by the ligand occupation of the second receptor [13], and even by morphologic studies like high-resolution two-dimensional crystal structures [14] and atomic force microscopy [15]. Another novel approach called bimolecular fluorescence complementation (BiFC) has been utilized to demonstrate receptor dimerization by coexpressing the non-fluorescent halves of yellow fluorescent protein (YFP) attached to receptor constructs and monitoring the formation of functionally intact YFP [16].

Functional complementation is another unique and important methodological approach. In this, a structurally and functionally deficient form of a receptor is corrected by the expression of another functionally deficient receptor that has distinct structural defects, such that the association of the two receptors can contribute all the necessary structural domains to yield a fully functional unit [17]. This was first utilized to prove the possibility of "crossed-domain dimerization." While there is little doubt that this can occur under decidedly unique experimental conditions, with high levels of expression of complementary defective receptor constructs, sufficient data have not definitively established the relevance of this type of GPCR dimerization in normal physiology. Rather, most studies in recent years have focused on the association of intact receptors having complete heptahelical bundles with each other [18].

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