Interaction of Prokineticin Receptors with Accessory Proteins: History
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Subjects: Neurosciences
Contributor:
Roberta Lattanzi
,
Rossella Miele

G protein-coupled receptors (GPCRs) are transmembrane proteins that mediate the intracellular pathway of signals not only through heterotrimeric GTP-binding proteins (G proteins) but also through their associations with a variety of additional partner proteins. Prokineticin receptors 1 (PKR1) and 2 (PKR2) are new members of the GPCRs whose ligands are the novel chemokines prokineticin 1 (PK1) and prokineticin 2 (PK2). The multiplicity of G proteins coupled to PKRs, the ability of PKR2 to heterodimerize, the interaction of PKR2 with accessory proteins, and the existence of alternative splice isoforms of PKR2/PK2 explain the complexity of the system in the signal transduction pathway and, consequently, in the modulation of various physiological and pathological functions. Knowledge of these mechanisms provides the basis for the development of targeted drugs with therapeutic efficacy in PK-dependent diseases.

  • G protein-coupled receptors
  • accessory proteins
  • prokineticins
  • prokineticin receptors
G protein-coupled receptors (GPCRs) are a family of transmembrane proteins that play a fundamental role in various physiological and pathophysiological processes in humans. For this reason, they are pharmacological targets for the development of new drugs with therapeutic efficacy in many conditions such as chronic pain, inflammation, neurodegenerative diseases, diabetes, stress, and osteoporosis. A substantial number of pharmaceutical drugs have GPCRs as a target, further highlighting their importance in population health [1].
Prokineticin receptors 1 (PKR1) and 2 (PKR2) belong to the family of A-GPCRs and are particularly similar to neuropeptide Y receptors (NPY). They show high sequence identity and greater sequence variability in their N-terminal region [2][3][4]. Prokineticin receptors are widely distributed in all districts: PKR1 is predominantly expressed in peripheral organs and tissues and in the peripheral nervous system (PNS), whereas PKR2 is mainly expressed in the central nervous system (CNS) [5]. In the rat hippocampus, a PKR2 splice variant has been identified that lacks the second exon and results in a protein that does not consist of the seven transmembrane domains typical of GPCRs, but of only four transmembrane elements, and is therefore called TM 4–7 [6].
Prokineticin receptors bind two ligands capable of triggering contraction of the guinea pig ileum and are therefore termed prokineticin 1 (PK1) and prokineticin 2 (PK2) [7]. The prokineticin system is found throughout the evolutionary scale. Orthologs of PK2 were first identified in amphibians [8] and reptiles [9] and then in fish and mammals [7].
PK1 is also known as EG-VEGF (endocrine gland-derived vascular endothelial growth factor) because it can induce proliferation, migration, and fenestration in the endothelial cells of steroid-synthesising glands [10][11]. PK2 is also called mammalian Bv8 (mBv8) [7][12] because it is the orthologue of the Bv8 protein isolated from the skin secretion of the frog Bombina variegata [8].
In humans and mice, the Pk2 gene, consisting of four exons [13], generates three distinct transcripts encoding PK2, PK2L, and PK2C through alternative splicing. PK2L, a long form of PK2 due to the presence of 21 additive basic amino acids, is inactive but rapidly cleaved to PK2β, a more PKR1-selective ligand [14][15]. The PK2C isoform is expressed in the mouse’s central nervous system, particularly in the hippocampus and spinal cord, and is encoded by exon 1 and exon 4 [16]. All proteins are also called AVITGA proteins [17] because they share a common and conserved N-terminal sequence consisting of alanine, valine, isoleucine, tryptophan, glycine, and alanine. The prokineticins are small GPCR ligands with ten cysteine residues forming five disulfide bridges. They act as chemotactic and immunomodulatory factors and are classified as chemokines [18].
Prokineticins bind to PKRs and the ligand/receptor complex undergoes conformational changes that induce activation of intracellular effectors (G-proteins or Arrestins) and cellular responses through various signal transduction pathways. PKRs activate various intracellular signaling pathways in different cell types by coupling to Gq, Gi, and Gs [5][12][19].
The specific functional properties of GPCRs such as ligand binding, activation, and trafficking can be modulated by their dimerisation [20]. PKR2, like several GPCRs, is capable of forming homodimers. By generating PKR2 mutants, it has been shown that dimerization occurs through interactions between transmembrane domains (TMs), particularly TM5, via a domain swapping mechanism. It has been shown that the co-expression of binding-deficient and signal-deficient forms of PKR2 can restore receptor functionality [5][12].
Evidence suggests that the interaction between prokineticins and prokineticin receptors follows the accepted model of chemokine/chemokine receptor interaction. This is a two-sided model involving a first interaction (site 1) that is critical for specificity and consists of the recognition of the N-terminus of the chemokine by the extracellular loop 2 (ECL2) of the receptor. The second interaction (site 2), which is responsible for the complex conformational changes that trigger transduction of the intracellular signaling pathway, results from the chemokine N-terminus entering the orthosteric transmembrane (TM)-binding site pocket of the GPCR [21].
Site 1 of prokineticin receptors was characterized, demonstrating the essential role of the extracellular surface of PKRs, particularly extracellular loop 2 (ECL2), in the binding sites for endogenous prokineticin ligands. Analysis of the PKR2 Q210R mutation, in which the glutamine at position 210 is replaced by arginine, demonstrated that the ECL2 glutamine residue is essential for PK2 binding in patients with Kallmann syndrome (KS) [22].
Using amber codon suppression technology, it has been demonstrated that Tryptophan at position 212 of PKR2 also plays a critical role in PKS ligand binding [23].
Computational analysis identified the PKRs orthosteric TM–binding site 2 and showed that, in humans, this site is almost identical in both PKR1 and PKR2. The only difference concerns the valine residue (V) 207 in PKR1, which is substituted by phenylalanine (F) 198 in PKR2. This allosteric transmembrane site also binds small non-peptide agonists and synthetic small non-peptide ligands that act as PKR antagonists [24][25][26] (Figure 1).
Figure 1. Schematic representation of PKR2 topology. Created in BioRender.com.
Endogenous prokineticin ligands bind to PKRs via two “hot spots” consisting of the region comprising the amino acid sequence AVITG and the conserved hydrophobic amino acid residue tryptophan at position 24 (Trp24). This is exposed on the surface of prokineticins and is critical for their PKR affinity and activity. The substitution of tryptophan 24 (Trp 24) with Alanine (Ala) produces a Bv8 analog called (Ala24-Bv8) that, when injected at high doses in both mice and rats, similarly to Bv8, acts as an agonist, inducing deep and long-lasting thermal hyperalgesia and tactile allodynia. Conversely, when administered at low, ineffective doses, it behaves like PKR-receptor antagonists and reverts Bv8-induced hyperalgesia and allodynia [12][27].
PKR2 is known to bind not only endogenous ligands but also the pathogenic parasite Trypanosoma cruzi, facilitating its invasion and infiltration into mammalian host cells. PKR2 specifically recognizes Tc85, a Trypanosoma glycoprotein that belongs to the trans-sialidase family [28]. The LamG domain of Tc85 activates PKR2 and induces the activation of ERK, NFAT, and STAT3 in CHO mammalian cells and in mouse spinal ganglion explants [28].
Prokineticins and prokineticin receptors are expressed in a variety of organs, including the central and peripheral nervous systems, heart, ovaries, testes, placenta, adrenal cortex, peripheral blood cells, and bone marrow. They are involved in a number of physiological functions such as hormone secretion and reproduction, angiogenesis, neurogenesis, the regulation of circadian rhythm, and the modulation of food intake and drinking. They are involved in pathological conditions such as pain, cancer, obesity, neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease, and genetic disorders such as Kallmann syndrome [5][12][29][30][31][32][33].
The different biological effects induced by the prokinetic system in various tissues is due to the multiplicity of G-proteins coupled to PKRs together with the alternative splicing isoforms of PK2 and PKR2 and the dimerization of PKRs.
For a long time, it was believed that the signal mediated by GPCRs depended exclusively on G proteins, which in turn were able to activate or inhibit downstream effectors, and that the variability of the response was due to the ability to couple different G proteins. Although the central role of G proteins is still recognized, it is now clear that GPCRs rely on associations with a variety of additional protein partners to complete their cycle. Accessory proteins that interact with GPCRs modulate cell signaling and receptor expression and/or pharmacological profiles.
In this review, we analyze how accessory proteins can control the exact synthesis of the PKRs, stabilize them on the cell membrane, modulate the ligand binding and the intracellular signal transduction pathway, and induce receptor desensitization and endocytosis.
Understanding the role of these PKR accessory proteins is important because they may be a target for drug development with therapeutic efficacy in PK-dependent diseases.

This entry is adapted from the peer-reviewed paper 10.3390/encyclopedia3040107

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