Dimerization of G-Protein-Coupled Receptors

Abstract

The evolutionary trace (ET) method, a data mining approach for determining significant levels of amino acid conservation, has been applied to over 700 aligned G-protein-coupled receptor (GPCR) sequences. The method predicted the occurrence of functionally important clusters of residues on the external faces of helices 5 and 6 for each family or subfamily of receptors; similar clusters were observed on helices 2 and 3. The probability that these clusters are not random was determined using Monte Carlo techniques. The cluster on helices 5 and 6 is consistent with both 5,6-contact and 5,6-domain swapped dimer formation; the possible equivalence of these two types of dimer is discussed because this relates to activation by homo- and heterodimers. The observation of a functionally important cluster of residues on helices 2 and 3 is novel, and some possible interpretations are given, including heterodimerization and oligomerization. The application of the evolutionary trace method to 113 aligned G-protein sequences resulted in the identification of two functional sites. One large, well-defined site is clearly identified with adenyl cyclase, beta/gamma and regulator of G-protein signaling (RGS) binding. The other G-protein functional site, which extends from the ras-like domain onto the helical domain, has the correct size and electrostatic properties for GPCR dimer binding. The implications of these results are discussed in terms of the conformational changes required in the G-protein for activation by a receptor dimer. Further, the implications of GPCR dimerization for medicinal chemistry are discussed in the context of these ET results.

Synthetic Multivalent Ligands as Probes of Signal Transduction

“…[49] For example, helix-mediated multimerization is proposed to mediate the dimerization and oligomerization of GPCRs (Figure 2). [38,50,51] Evidence for the functional importance of these interactions is suggested by engineering disrupting mutations in the proposed contact sites or by adding isolated transmembrane helices to GPCRs. [37,52,53] Although their effects on receptor oligomerization are not yet well-established, these manipulations modulate signaling; presumably, they disrupt receptor-receptor contacts.…”

Regulation of the Coupling to Different G Proteins of Rat Corticotropin-releasing Factor Receptor Type 1 in Human Embryonic Kidney 293 Cells

“…It might be speculated that there is an allosteric site the occupation of which at high agonist concentrations negatively modulates the cyclase stimulation by the orthosteric receptor binding site. Evidence has accumulated that receptor proteins may form dimers especially when overexpressed and that the second binding site to be occupied in the dimer may negatively modulate the response of the active receptor state (27). It could be possible that rCRFR1 in the HEK cells forms dimers, resulting in allosteric inhibition of adenylate cyclase at high ligand concentrations.…”

Homodimerization of Neuropeptide Y Receptors Investigated by Fluorescence Resonance Energy Transfer in Living Cells

“…NPY receptors belong to the rhodopsin-like superfamily of Gprotein-coupled receptors (35). So far five distinct NPY receptors have been cloned (Y 1 , Y 2 , Y 4 , Y 5 , and y 6 ) (36). Their activation results in the inhibition of adenylyl cyclase and in an increase in intracellular calcium concentration.…”

“…To investigate homodimerization of NPY receptors by FRET, we generated fusion proteins of the hY 1 -, hY 2 -, or hY 5 -receptor sequence tagged at their carboxyl terminus to the green, cyan, yellow, or red fluorescent protein (GFP, CFP, YFP, DSRed), respectively. The advantage of these proteins is that they have the suited properties to be used as FRET pairs (44 -46).…”