Mutations of neighboring polar residues on the second transmembrane helix disrupt signaling by the parathyroid hormone receptor.
Site-directed mutagenesis was used to assess the role of transmembrane (TM)-charged amino acids in the expression and function of the G protein-coupled receptor for PTH and PTH-related protein (PTHrP). Charged residues that are conserved in the TM regions of most or all members of the PTH/secretin receptor subfamily were targeted. Four mutants (E296A, R337A, H414A, and E459K) displayed properties similar to the wild type PTH/PTHrP receptor with respect to agonist binding and stimulation of adenylyl cyclase when expressed in COS-7 cells. Several mutations, all in TM II, produced receptors that signaled extremely poorly. Mutation of three residues (227S, 230R, and 233S), predicted to be aligned on one helical face of TM II, displayed a similar phenotype: markedly blunted adenylyl cyclase activity in response to PTH (20-30% of the wild type response) and a lower binding affinity for agonist, with no reduction in cell surface receptor expression. These results suggest that TM II contains a polar face that is involved in TM signaling by the PTH/PTHrP receptor. Two of these mutations were made at the corresponding sites in the secretin receptor, and a similar reduction in secretin-stimulated adenylyl cyclase activity was observed. Thus this region of TM II may participate in a mechanism of TM signal transduction that is shared by the PTH/secretin sub-family of G protein-coupled receptors.
Turner
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PR
Endocrine Unit, Veterans Affairs Medical Center, San Francisco California, 94121, USA.
Bambino
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T
Nissenson
R A
RA
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010281:Parathyroid Hormone / Q000235:genetics; D044162:Parathyroid Hormone-Related Protein; D011506:Proteins / Q000235:genetics; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone / Q000235:genetics*; D015398:Signal Transduction
D010281:Parathyroid Hormone; D044162:Parathyroid Hormone-Related Protein; D011506:Proteins; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone; D019204:GTP-Binding Proteins