GPCR interactions with cytoplasmic proteins

GPCRs have been found to interact with increasing numbers of cytoplasmic proteins (Table 17). The physiological significance of these interactions is often still unclear.

RCP is a small (20 kDa) intracellular peripheral membrane protein. Unlike the RAMP proteins, RCP does not act as a chaperone, but rather plays a role in coupling these receptors to downstream signalling molecules. In this respect, the new model for CGRP/adrenomedullin signalling requires a complex of three proteins to confer full receptor-mediated function: CRLR, RAMP1 or 2 and RCP (Figure 152).

Whereas G proteins are widely recognized to play a central role in the transfer of information between GPCRs and cellular effectors, several recent findings suggest that non-G proteins can also convey this information. To deal with these new findings, it has been suggested that 'G protein-coupled receptors' should no longer be called such,

Table 17 GPCR interaction with cytoplasmic proteins. Reprinted from Cellular Signalling, 14, Brady, A. E. and Limbird, L. E., G protein-coupled receptor interacting proteins: Emerging roles in localization and signal transduction, 297-309. Copyright (2002), with permission from Elsevier.

GPCR interacting proteins

Functional role(s) implicated

P2 receptor


Regulation of brush border NHE3 activity;

sorting selection of surface recycling versus

lysosomal targeting after agonist-evoked


5HT2C receptor


Not known, although co-localized in native



SSTRIP (member

At postsynaptic densities; critical for PSD

of Shank/Spank/

localization and/or SSTR2 signalling

synamon family)

D2 receptor


Function unknown

a2 receptor (A,B,C)


Agonist-enhanced co-IP in heterologous cells;

function in native cells unknown

AT1a receptor


Interaction via AT1a receptor C-terminus;

ATRAP overexpression inhibits

angiotensin-stimulated PLC, smooth

muscle cell growth; increases AT1a

receptor internalization



Trimer permits CGRP receptor (CLR/

RAMP1) and adrenomedullin receptor

(CCRLR/RAMP2) to couple to activation

of adenylyl cyclase

mGluR5 mGluR1a


Regulation of mGluR availability for

interaction with Homer-2 and -3 and

their interacting proteins at postsynaptic

densities; Homer-1a expression is

upregulated by seizure-induced activation

of the hippocampus

mGluR5, mGluR1a


Retention of receptors in ER

mGluR5, mGluR1a


Clustering of receptors at cell surface;

enriched dendritic localization in rat

hippocampal neurons

P2 receptor

AKAP250 (gravin)

Regulates receptor (gravin) sequestration and

resensitization; associates with P2 receptor

during agonist-elicited endocytosis

P2 receptor

AKAP79 (rat

Enhances P2 receptor phosphorylation by



D2 R (long, short


Clustering of receptors at cell surface;


(Filamin A)

increased efficiency of coupling of receptor

to inhibition of adenylyl cyclase

a2 receptor

14-3-3 £

Function unknown; competed for by

phosphorylated Raf peptide


14-3-3 £ and n

Not known

a2 /p2 receptors


Subtle (15%) decrease in P2 receptor-

stimulated adenylyl cyclase


Activity; colocalisation in surface blebs

created by overexpression, but not at points

of cell-cell contact

Figure 154 P2-adrenergic receptor-mediated stimulation of the Na+/H+ exchanger type 3 in the plasma membrane. Reprinted from Trends in Pharmacological Science, 19, Schwartz, T. W., IJzerman, A. P., Principles of agonism: undressing efficacy, 433-436. Copyright (1998) with permission from Elsevier.

but that their denomination as 'heptahelical receptors' or '7TM receptors' would be more appropriate.

Examples of alternative signalling pathways are:

• P2-adrenergic receptors also activate a Na+/H+ exchanger type 3 (NHE3) in the cell plasma membrane independently of Gs (Figure 154). To this end, the activated receptor directly associates with the Na+/H+ exchanger regulatory factor (NHERF). Activated NHERF then activates the Na+/H+ exchanger, resulting in increased sodium reabsorption in the proximal tubule of the kidney. NHERF interacts through its 'PDZ domain' with the C-terminal tail of the P2 receptor. The affected receptor region is not involved in the recognition of G proteins since a Leu to Ala mutation of the final residue of the receptor abolishes its interaction with NHERF without altering its ability to activate the adenyl cyclase system. Besides NHERF, GPCRs have been shown to interact with a number of PDZ domain-containing proteins, such as spinophilin.

• Recently, some GPCRs, including M3 muscarinic acetylcholine receptor and Hi histamine receptor, have been suggested to stimulate the membrane-associated phospholipase D enzyme in an Arf- and RhoA-dependent manner, without the involvement of G proteins. Receptors of this type contain an AsnProXXTyr motif in their TM7 domain that is able to form complexes with Arf and RhoA, leading to the activation of phospholipase D.

• Agonist-stimulated P3-adrenergic receptors stimulate the ERK/MAP kinase cascade by directly binding to c-Src. This interaction requires proline-rich motifs (PXXP) in the endo3 loop and the C-terminal end of the receptors and the SH3 domain of c-Src. By contrast, the interaction between c-Src and the P2-adrenoceptor requires P-arrestin as a scaffold.

GPCRs also interact with cytoplasmic proteins with a 'scaffolding' function (Wang and Limbird, 2002):

• Actin-binding protein-280 (ABP-280), also known as filamin A, is an abundant cytoplasmic protein that may act as a scaffold to anchor a number of different molecules at the cytoskeleton. ABP-280 helps D2 receptor clustering (micro-compartmentalization) at the plasma membrane and, by enhancing the efficiency of coupling to effectors, it facilitates D2 receptor signalling. This may explain the increased sensitivity of presynaptic D2 to agonist stimulation (when compared to postsynaptic D2 receptors).

• Spinophilin (120 kDa), endo3 (PDZ domain-containing) F-actin-binding protein, has been found to interact with the loop of both D2 and a2-adrenergic receptors. It may act as a scaffolding protein that links signalling proteins to microdomains at the cell surface (e.g. a2 receptor retention at the basolateral surface of polarized epithelial cells).

• 14-3-3 proteins (31 kDa) are predominantly cytosolic and expressed as seven known mammalian isoforms. They recognize a phosphorylated serine/threonine motif (RSxSxP) of molecules like a2-adrenergic receptors and modulate their function. 14-3-3 proteins exist as dimers, thus allowing them to act as a scaffold for coordinating signal transduction.

The interactions of the a2-adrenergic receptor endo3 loop with spinophilin, 14-3-3Z, and arrestin 3 are capable of competing with each other (Figure 155). Relative affinities of these peptides for the endo 3 loop of the receptor can be tested with a pull-down assay where a glutathione-S-transferase (GST)-spinophilin fusion protein is present at the surface of (glutathione) GSH-agarose. This is incubated with the 35S-labelled endo3 loop of the a2A-adrenergic receptor and increasing concentrations of competing peptides. At the end, the amount of 35S present on the beads is measured and the relative binding affinity of the competing peptides can be evaluated.

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