Identification of an Essential Amino Acid Motif within the C Terminus of the Pituitary Adenylate Cyclase-activating Polypeptide Type I Receptor That Is Critical for Signal Transduction but Not for Receptor Internalization

Abstract

The pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 (PAC1) receptor is a G proteincoupled receptor and class II receptor member. The receptor domains critical for signaling are unknown. To explore the role of the C terminus, truncations of 63 residues (Tr406), 53 residues (Tr416), 49 residues (Tr420), 44 residues (Tr424), and 37 residues (Tr433) were constructed and expressed in NIH/3T3 cells, and immunofluorescence, radioligand binding, adenylyl cyclase (AC) and phospholipase C (PLC) assays were performed. 125 It has been previously demonstrated for receptors belonging to the class I heptahelical receptor family that specific intracellular amino acid residues are critical for internalization and coupling to guanine nucleotide-binding proteins (G proteins). These cytoplasmic domains have been partially characterized for the ␣-and ␤-adrenergic receptors (1, 2), the muscarinic receptors (3), and for rhodopsin (4). Studies of the ␤ 2 -adrenergic (2) and muscarinic receptors (3) have suggested that substitution, deletion, and/or insertion of residues in the third intracellular loop affect the ability of these receptors to couple to G proteins.At present, the role of the cytoplasmic domains for members of the class II family for receptor coupling to intracellular G proteins has not been fully explored. Based on studies with receptors belonging to the class I receptor family, we speculated that amino acids in either the third intracellular loop or the C terminus of the pituitary adenylate cyclase-activating polypeptide receptor (PAC1), 1 may be important for coupling to G proteins (5). Furthermore, the C terminus has been proposed to be necessary for the formation of a functional receptor-G protein complex and to result in heterodimerization for some GPCRs such as type B receptor for GABA and GABA B -like orphan receptor (6).Pituitary adenylate cyclase-activating polypeptide (PACAP) hormone was originally isolated from bovine hypothalamus and is one of the most recently identified peptides in the VIP/ secretin/glucagon family (7). The cloning of the rat and human PAC1 cDNA and genes identified a receptor protein composed of ϳ468 amino acids containing seven transmembrane domains that are characteristic of G protein-coupled receptors (5, 8). The PAC1 belongs to the class II GPCR superfamily that includes VPAC1 (the classical VIP1 receptor), VPAC2 (VIP2 receptor), secretin, glucagon, and calcitonin receptors (9); these molecules can be distinguished on the basis of their relative affinities for the agonists PACAP-27, PACAP-38, VIP, and helodermin (10). The cloning of the rat and human PAC1 gene have identified two unique exons that can be alternatively spliced yielding different third intracellular loops. The four major splice variants are named Null, Hip, Hop,8), and they have been shown to be differentially expressed in different tissues and are able to couple with different efficacies and potencies to phospholipase C (PLC) and adenylyl cyclase (AC) (5,8). Therefore, the expression of splice varian...

Regulatory Properties of α1B-Adrenergic Receptors Defective in Coupling to Phosphoinositide Hydrolysis

“…Studies of additional mutations, including smaller deletions as well as substitution mutations, will be required to more precisely identify the roles of this region of the receptor and of individual amino acid residues in both G protein coupling and endocytosis. Several recent studies with other receptors have succeeded in separating the structural domains involved in internalization from those involved in G protein activation for various G q -coupled receptors (Hunyady et al, 1994;Bhowmick et al, 1998;Lyu et al, 2000). Of greatest interest in relation to our data is a recent study of the regulatory properties of the constitutively active D142A-mutated ␣ 1B AR, which was able to activate G q and PI hydrolysis but was defective in both agonist-induced receptor phosphorylation and internalization .…”

“…Recent studies with other G q -coupled receptors have also provided evidence that G q coupling and PI hydrolysis are not required for receptor internalization. These include studies with mutated receptors for AT 1 angiotensin receptors (Hunyady et al, 1994), ET A endothelin receptors (Bhowmick et al, 1998), and pituitary adenylyl cyclase-activating protein type 1 receptors (Lyu et al, 2000) as well as studies demonstrating thyrotropin-releasing hormone receptor internalization in cells lacking G q and G 11 (Yu and Hinkle, 1999). The data presented here appear to conflict with other evidence that the PKC inhibitor staurosporine is effective at inhibiting ␣ 1B AR internalization (Fonseca et al, 1995;Zhu et al, 1996;Awaji et al, 1998).…”

Serine 447 in the Carboxyl Tail of Human VPAC1 Receptor Is Crucial for Agonist-Induced Desensitization but Not Internalization of the Receptor

“…On the one hand, the cells were lysed with 500 l of 0.5 M NaOH at 25°C and the measured radioactivity was used to estimate specific 125 I-VIP binding to the cells. On the other hand, bound 125 I-VIP at the cell surface was stripped by an incubation with 0.5 M KSCN solution at room temperature during 10 min as described previously (Lyu et al, 2000). The amount of specifically internalized 125 I-VIP was then estimated and expressed as a percentage of 125 I-VIP specifically bound to CHO cells in function of time.…”

Identification of Cytoplasmic Domains of hVPAC1 Receptor Required for Activation of Adenylyl Cyclase

“…Indeed, some class I receptors use the proximal C-terminal tail for G protein coupling (37) whereas others activate G protein even though they are not equipped with a C-terminal tail (38). With regard to class II GPCRs, there is evidence for a role of the C-terminal tail in signal transduction for the porcine calcitonin receptor (39) and the human PAC1 receptor for PACAP (40). The C-terminal tail of the rat glucagon receptor was claimed not to be necessary for adenylyl cyclase coupling (41).…”