Dopamine D₄ receptor oligomerization – contribution to receptor biogenesis

Abstract

Dopamine D 4 receptors (D 4 Rs) are G protein-coupled receptors that play a role in attention and cognition. In the present study, we investigated the dimerization properties of this receptor. Western blot analysis of the human D 4.2 R, D 4.4 R and D 4.7 R revealed the presence of higher molecular weight immunoreactive bands, which might indicate the formation of receptor dimers and multimers. Homo-and heterodimerization of the receptors was confirmed by co-immunoprecipitation and bioluminescence resonance energy transfer studies. Although dimerization of a large number of G protein-coupled receptors has been described, the functional importance often remains to be elucidated. Folding efficiency is rate-limiting for D 4 R biogenesis and quality control in the endoplasmic reticulum plays an important role for D 4 R maturation. Co-immunoprecipitation and immunofluorescence microscopy studies using wild-type and a nonfunctional D 4.4 R folding mutant show that oligomerization occurs in the endoplasmic reticulum and that this plays a role in the biogenesis and cell surface targeting of the D 4 R. The different polymorphic repeat variants of the D 4 R display differential sensitivity to the chaperone effect. In the present study, we show that this is also reflected by bioluminescence resonance energy transfer saturation assays, suggesting that the polymorphic repeat variants have different relative affinities to form homo-and heterodimers. In summary, we conclude that D 4 Rs form oligomers with different affinities and that dimerization plays a role in receptor biogenesis.Structured digital abstract l D4.4R physically interacts with D4.2R by anti tag coimmunoprecipitation (View interaction) l D4.xR physically interacts with D4.4R by anti tag coimmunoprecipitation (View interaction) l D4.xR and D4.xR physically interact by bioluminescence resonance energy transfer (View interaction)Abbreviations BRET, bioluminescence resonance energy transfer; CHO, Chinese hamster ovary; DAPI, 4¢,6-diamidino-2-phenylindole; D n R, dopamine D n receptor; ER, endoplasmic reticulum; GPCR, G protein-coupled receptor; HRP, horseradish peroxidase; MP, milk powder; YFP, yellow fluorescent protein.

G protein-coupled receptor-receptor interactions give integrative dynamics to intercellular communication

“…Mutagenesis studies targeting the putative interaction interface of β 2 -adrenergic receptor showed that inhibiting β 2 dimerization caused its retention in the ER, thus supporting the hypothesis of receptor oligomerization in this cellular compartment (Salahpour et al, 2004). Other examples encompass the D 4 receptors (Van Craenenbroeck et al, 2011), GABA B receptors (MargetaMitrovic et al, 2000), and 5-HT 2C receptors (Herrick-Davis et al, 2006).…”

The role of D4 receptor gene exon III polymorphisms in shaping human altruism and prosocial behavior

“…Functional significance of these repeats has been suggested in many studies (Van Tol et al, 1992; Asghari et al, 1994, 1995; Schoots and Van Tol, 2003; Van Craenenbroeck et al, 2005, 2011). For example, the 7R has been linked to suppressed DRD4 expression in vitro (Schoots and Van Tol, 2003).…”

Receptor-receptor interactions in heteroreceptor complexes: a new principle in biology. Focus on their role in learning and memory

“…Besides interacting with numerous ER-resident folding assistants GPCRs can self associate in the ER forming GPCR homo-and heteromers which under some circumstances might have a chaperone-like activity defining the receptor's export profile [62,65,66]. Alternatively, GPCRs can interact in this organelle with accessory proteins (e.g., RAMP, MRAP, RanBP2, MRAP, etc.)…”