PAC1 (PACAP Receptor) Receptor Splice Variants and Effects on PAC1 Function

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Description of the PAC1 (PACAP receptor) receptor splice variants and the effects they have on PAC1 function.

 

INTRODUCTION

G-protein-coupled receptors (GPCRs) represent the largest family of membrane-associated proteins mediating physiological responses in vertebrates by means of controlling metabolic, neural, and developmental functions (1). They are expressed in almost all types of tissues and respond to diverse extracellular signals like hormones, neurotransmitters, pheromones, lipids, and other proteins. They are the target for 30 – 40% of all marketed drugs. The GPCRs consist of seven-transmembrane domains with 3 intracellular and 3 extracellular loops. The N-terminus lies outside the cell and is involved in the diverse ligand recognition process together with the 3 extracellular loops. Three intracellular loops and a C-terminal domain are involved with the transduction of the signal inside the cell and nucleus. When a ligand binds to extracellular domain (either the extracellular loop or the N-terminus), it induces conformational changes in the transmembrane and intracellular domains of the receptor resulting binding of G-proteins. G-proteins in turn regulate the activity of effector molecules inside the cell, causing the activation of secondary messengers such as cyclic AMP (cAMP), inositol-triphosphate (IP3), and diacylglycerol leading to the initiation of distinct signalling pathways (2). Besides the diversity in the ligand binding and transducing effectors further diversity in the GPCRs can be obtained through the alternative splicing of different exons of the gene encoding the receptor. Accumulating evidence suggests that presence of alternative splicing substantially adds to the functional diversity to the human genome (3). Alternative splicing allows for essential and precisely regulated differences in tissue-specific expression as well as in ligand binding, internalization, and intracellular signalling properties of the GPCRs (3; 4).

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The PAC1 receptor belongs to the class B family of GPCRs and has a large extra-cellular N-terminal domain. It is activated by the neuropeptide Pituitary adenylate cyclase-activating peptide (PACAP). There are two variants of this peptide PACAP38 and PACAP27. This essay describes the splice variants of the PAC1 receptor and the effects they have on its function. The alternative splicing of this receptor leads to different physiological outcomes.

The PAC1 receptor and alternative splicing

The activated PAC1 receptor can signal through multiple pathways. It can signal through cAMP by coupling to the

Gαs

G-protein or through

Ca2+ 

or Inositol trisphosphate (IP3) by coupling to the

Gαq

 G-protein. The PAC1 receptor plays a pivotal role in spatiotemporal regulation of cell proliferation, differentiation and survival during development as well as in the regulation of synthesis and release of neuroendocrine hormones (5). The human PAC1 receptor is coded by gene ADCYAP1R1 which is organized into 18 exons that undergo extensive alternative splicing giving rise to different splice variants (5; 6). Ten of these exons are constitutively expressed (exons 2,3,7–13,18), whereas the rest (exons 4–6, 14,15, and possibly 16,17) are regulated by the alternative splicing (5). The N-terminal part of PAC1 receptor is encoded by exons 2-6. The seven transmembrane domain including extracellular and intracellular loops is encoded by exons 7-17. The C-terminal cytoplasmic tail including the 3′ untranslated region is encoded by the exon 18 (5). A diagrammatic representation of the PAC1 gene can be seen in the figure1 below. The alternative splicing of the PAC1 receptor can be divided into four types (5) (i) N-terminal domain splice variants altering the ligand-binding specificity and affinity (ii) intracellular loop 3 (IC3) splice variants affecting binding of G-protein and / or interaction with other intracellular signalling proteins. (iii) transmembrane domains 2 and 4 splice variants affecting the receptors heterodimerization and intracellular transport. (iv) 5′ untranslated region splice variants that affect mRNA expression dynamics (5). The PAC1 receptor that has the 468 amino acids with full length N-terminal domain and without the intracellular domain 3 insertion cassettes is called the PAC1-null and is normally used as a reference to compare activity of the other splice variants (5; 7).

N-TERMINAL Splice VAriants

The N-terminal splice variants of the receptor are generated by alternative splicing of (i) exon 5 leading to a deletion of 7 amino acids (PAC1- 5) (ii) exons 5–6 leading to a deletion of 21 amino acids (PAC1-short or PAC1- 5,6) (iii) exons 4–6 leading to a deletion of 57 amino acids (PAC1-very short or PAC1- 4,5,6) (5; 8). In rat, another variant is found that has an insertion of 24 amino acids caused by splicing of a novel exon 3a located between exons 3 and 4 (PAC1-3a) (5). N-terminal splicing isoforms of PAC1 display alterations in affinity and selectivity of ligand-binding and coupling to second messengers.

INTRACELLULAR LOOP Splice VAriants

These splice variants are generated by the inclusion of exons 14 or 15 and encodes for 28 extra amino acids. PAC1-hip splice variants include the exon 14 and PAC1-hop splice variants include the exon 15. PAC1-hip-hop include both exon 14 and 15 (7). In rat there are another two variants of the exon 15 which encode 28 (hop1) or 27 (hop2) amino acids using two adjacent 5′ splice acceptor sites (7; 9). Additionally, another splice variant is found in rat that is formed by a C-terminal deletion of 193 nucleotides that includes two amino acids that are essential for G-protein recognition. This receptor is named PAC1-hop1-novel (5; 10). In human, hip and hop variants are also referred to as SV1 and SV2 and hip-hop variant as SV3 (5). Intracellular splicing isoforms of PAC1 can profoundly influence ligand association and activation of the PAC1 receptor and alter intracellular signal transduction (7).

TRANSMEMBRANE DOMAIN Splice VAriants

The exact molecular mechanism underlying these variations remains unclear (5). A PAC1 variant, in rat, has amino acids deletion/substitution in the transmembrane 4 domain and one amino acid substitutions in the N-terminal (D136N) and one in transmembrane 2 domain (N190D). The transmembrane 4 domain of the receptor is usually involved in homo and hetero dimerization of the receptors and associations with the RAMP proteins, and GPCR kinases. Thus, suggesting that splice variants of transmembrane 4 domain may affect heterodimerization and intracellular transport (5; 11). Currently there is insufficient data regarding this type of variant to make any conclusion about pharmacological or physiological significance (9).

5′ untranslated region splice variants

The nature and function of the alternative splicing of 5′ untranslated region in class B GPCR receptors in humans is yet to be determined (9). But some alternative splicing events have been identified in the rat PAC1 gene for exons located at the 5′ untranslated region (5; 6). These include different alternative usage of exons located upstream to the ATG translation start codon (5). Variations in the 5′ untranslated region sequences may play a role in the regulation of mRNA expression (5).

effect of various splice variants on LIGAND-BINDING

PAC1 displays a high-affinity for PACAP and low affinity to the vasoactive intestinal polypeptide (VIP) (12). But this changes for some of the splice variants of the PAC1. The C-terminal part of the ligand PACAP binds to the N-terminus of the PAC1 receptor and the N-terminal part of PACAP binds to the receptor’s extracellular loops (5). PAC1- 4,5,6, has a decreased affinity to PACAP27 and PACAP38 but its affinity toward VIP remains the same (5; 8). The binding affinities of the PAC1- 5 and PAC1- 5,6 splice variants appear to be unchanged for PACAP38, but PAC1- 5,6 shows an increased affinity toward VIP (5). The rat-specific PAC1-3a variants displays increased affinity towards PACAP38 but not PACAP27 (13). However, PACAP is still the better ligand for PAC1 when compared with VIP (5).

The PAC1-hop1 and PAC1-hip-hop1 splice variants retain similar binding properties to those of PAC1-null. But PAC1-hip and PAC1-hop2 variants show an increased affinity to VIP and a decreased affinity for PACAP peptides (5; 14).

Splice variants resulting from combined changes in the extracellular and intracellular domains show interesting results. A splice variant composed of PAC1- 5,6 and hop1, has similar binding properties for PACAP and VIP as PAC1-null but different binding properties for VIP as compared to PAC1- 5,6 alone (5; 7). Another combined splice variant composed of PAC1- 5 or PAC1- 5,6 with hip cassette shows different ligand binding properties to that of PAC1, PAC1- 5, or PAC1-hip alone (5; 7). Since transmembrane domain 6 is important for the binding of the ligand, an insertion in the intracellular loop 3 by the hip or hop cassettes can cause a conformational change in this domain thus affecting the binding of the ligands (5). Insertion of the hip cassette influences ligand activation and it functionally interacts with the full-length N-terminal domain. This gives a greater affinity for VIP but not PACAP-38 to the PAC1-hip with full-length N-terminal and not to the PAC1- 5,6 (7).

effect of splice variants on INTRACELLULAR SIGNAL TRANSDUCTION

Splice variants with modified or deleted intracellular 3, transmembrane 6 or C-terminal domains that are important for G-protein binding show a loss or reduction in the production of cAMP and IP3 eg PAC1-3a, PAC1- 4,5,6-hip, PAC1-hip-hop, PAC1- 5-hip, PAC1- 5,6-hip, PAC1-hop1-novel and PAC1- 5,6,14–17 (5). PAC1-hip and PAC1-hop1 show an increased coupling to cAMP and PLCβ, respectively (14).

The effects of PAC1 splice variants on the levels of cytoplasmic

Ca2+

were reported in the cases of PAC1-null, PAC1-hop1, PAC1-hop2, PAC1- 5,6 and PAC1-3a (5). PAC1-hop1 shows an elevation in

Ca2+

in comparison to PAC-null. PAC1-3a induces

Ca2+

accumulation through coupling to Gs/cAMP rather than Gq/PLCβ pathway (15). PAC1- 5,6 and PAC1- 5,6-hop1 exhibit PACAP induced cytoplasmic calcium elevation (5).

Both PAC1-null and PAC1-hop1proteins were also shown to signal through the non-canonical pathways by activating phospholipase D (PLD). Although PAC1-null activated PLD through Gq, PAC1-hop1 is able of activate PLD by directly binding to ADP-ribosylation factor (5).

CONCLUSION

Alternative splicing of the PAC1 receptor gene creates multiple functional proteins and variants that display a diversity in the receptor phenotype, ligand binding properties and altered intracellular signal transduction pathways. This allows the receptor to function differentially and also to be expressed differentially in different tissues of the body. Studies have shown that some splice variants are involved in the regulation of homeostasis in the body and adaptation during stressful encounters. Understanding the mechanism of signalling pathways of the various alternatively spliced variants of the receptor can help in the development of drugs that are better suited and efficiently targeted.

 

Figure 1 – A diagrammatic representation of PAC1 gene (ADCYAP1R1) Exons 2-18 encode the open reading frame. Exons that undergo alternative splicing are marked with the asterisk (5).

 

 

 

 

References

1. Seven transmembrane receptors: something old, something new. Lefkowitz, R.J. 2007, Acta Physiol (Oxf).

2. Diversity of G proteins in signal transduction. Simon, M.I., Strathmann, M.P. and Gautam, N. 252, 1991, Science, pp. 802-808.

3. Alternative Splicing of G Protein Coupled Receptors: Relevance to Pain Management. Oladosu, Folabomi A., Maixner, William and Nackley, Andrea G. August 2015, Mayo Foundation for Medical Education and Research.

4. Mu opioids and their receptors: evolution of a concept. GW, Pasternak and Y-X, Pan. 2013, Pharmacol Reviews.

5. Alternative splicing of the pituitary adenylate cyclase activating polypeptide receptor PAC1: mechanisms of fine tuning of brain activity. Blechman, Janna and Levkowitz, Gil . May 23, 2013, Frontiers in Endocrinology, Vol. 4.

6. Genomic organization of the rat pituitary adenylate cyclase activating polypeptide receptor gene. Alternative splicing within the 5’-untranslated region. Chatterjee, T.K., et al. 1997, The Journal of Biological Chemistry, Vol. 272.

7. Characterization of novel splice variants of the PAC1 receptor in human neuroblastoma cells: Consequences for signalling by VIP and PACAP. Lutz, E.M., et al. 2006, Molecular and cellular neurosciences.

8. The PACAP receptor: generation by alternative splicing of functional diversity among G protein-coupled receptors in nerve cells. Journot, L., et al. 1994, Cell Biology.

9. Consequences of splice variation on Secretin family G protein coupled. SG, Furness, et al. s.l. : British journal of pharmacology, 2012.

10. Pituitary adenylyl cyclase-activating polypeptide (PACAP) and its receptor (PAC1- R) in the cochlea: evidence for specific transcript expression of PAC1-R splice variants in rat micro dissected cochlear subfractions. Abu-Hamdan, M.D., et al. 2006, Neuro- science 140, 147–161.

11. Fine tuning of GPCR activity by receptor interacting proteins. Ritter, S.L. and Hall, R.A. 2009, Molecular Cell Biology 10, 819–830.

12. PAC1 receptors mediate pituitary adenylate cyclase-activating polypeptide and progesterone facilitated receptivity in female rats. Apostolakis, E.M., Riherd, D.N. and O’Malley, B.W. 2005, Molecular Endocrinology. 19, 2798–2811.

13. Novel alternatively spliced exon in the extracellular ligand-binding domain of the pituitary adenylate cyclase-activating polypeptide receptor (PAC1R) selectively increases ligand affinity and alters signal transduction coupling during spermatogenisis. Daniel, P.B., et al. 2001, The Journal of Biological Chemistry.

14. Differential signal transduction by five splice variants of the PACAP receptor. Spengler, D., et al. 1993, Nature 365, 170–175.

15. VIP provides cellular protection through a specific splice variant of the PACAP receptor: a new neuroprotection target. Pilzer, I. and Gozes, I. 2006 , Peptides 27, 2867–2876.

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